Methods for modulating bone mineralization

ABSTRACT

Antibodies to a novel family of TGF-β binding proteins are disclosed, for use in methods of altering bone density and bone mineralization, which methods are applicable to the treatment of diseases including osteopenia and osteoporosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed under 35 U.S.C. § 120 as a divisional of U.S.application Ser. No. 10/095,248, filed Mar. 7, 2002 and currentlypending, which is a continuation of U.S. application Ser. No.09/449,218, filed Nov. 24, 1999, now U.S. Pat. No. 6,395,511, whichclaims priority to U.S. Provisional Application No. 60/110,283 filedNov. 27, 1998, all of which are incorporated by reference in theirentirety.

TECHNICAL FIELD

The present invention relates generally to pharmaceutical products andmethods and, more specifically, to methods and compositions suitable forincreasing the mineral content of bone. Such compositions and methodsmay be utilized to treat a wide variety of conditions, including forexample, osteopenia, osteoporosis, fractures and other disorders inwhich low bone mineral density are a hallmark of the disease.

BACKGROUND OF THE INVENTION

Two or three distinct phases of changes to bone mass occur over the lifeof an individual (see Riggs, West J. Med. 154:63–77, 1991). The firstphase occurs in both men and women, and proceeds to attainment of a peakbone mass. This first phase is achieved through linear growth of theendochondral growth plates, and radial growth due to a rate ofperiosteal apposition. The second phase begins around age 30 fortrabecular bone (flat bones such as the vertebrae and pelvis) and aboutage 40 for cortical bone (e.g., long bones found in the limbs) andcontinues to old age. This phase is characterized by slow bone loss, andoccurs in both men and women. In women, a third phase of bone loss alsooccurs, most likely due to postmenopausal estrogen deficiencies. Duringthis phase alone, women may lose an additional 10% of bone mass from thecortical bone and 25% from the trabecular compartment (see Riggs,supra).

Loss of bone mineral content can be caused by a wide variety ofconditions, and may result in significant medical problems. For example,osteoporosis is a debilitating disease in humans characterized by markeddecreases in skeletal bone mass and mineral density, structuraldeterioration of bone including degradation of bone microarchitectureand corresponding increases in bone fragility and susceptibility tofracture in afflicted individuals. Osteoporosis in humans is preceded byclinical osteopenia (bone mineral density that is greater than onestandard deviation but less than 2.5 standard deviations below the meanvalue for young adult bone), a condition found in approximately 25million people in the United States. Another 7–8 million patients in theUnited States have been diagnosed with clinical osteoporosis (defined asbone mineral content greater than 2.5 standard deviations below that ofmature young adult bone). Osteoporosis is one of the most expensivediseases for the health care system, costing tens of billions of dollarsannually in the United States. In addition to health care-related costs,long-term residential care and lost working days add to the financialand social costs of this disease. Worldwide approximately 75 millionpeople are at risk for osteoporosis.

The frequency of osteoporosis in the human population increases withage, and among Caucasians is predominant in women (who comprise 80% ofthe osteoporosis patient pool in the United States). The increasedfragility and susceptibility to fracture of skeletal bone in the aged isaggravated by the greater risk of accidental falls in this population.More than 1.5 million osteoporosis-related bone fractures are reportedin the United States each year. Fractured hips, wrists, and vertebraeare among the most common injuries associated with osteoporosis. Hipfractures in particular are extremely uncomfortable and expensive forthe patient, and for women correlate with high rates of mortality andmorbidity.

Although osteoporosis has been defined as an increase in the risk offracture due to decreased bone mass, none of the presently availabletreatments for skeletal disorders can substantially increase the bonedensity of adults. There is a strong perception among all physiciansthat drugs are needed which could increase bone density in adults,particularly in the bones of the wrist, spinal column and hip that areat risk in osteopenia and osteoporosis.

Current strategies for the prevention of osteoporosis may offer somebenefit to individuals but cannot ensure resolution of the disease.These strategies include moderating physical activity (particularly inweight-bearing activities) with the onset of advanced age, includingadequate calcium in the diet, and avoiding consumption of productscontaining alcohol or tobacco. For patients presenting with clinicalosteopenia or osteoporosis, all current therapeutic drugs and strategiesare directed to reducing further loss of bone mass by inhibiting theprocess of bone absorption, a natural component of the bone remodelingprocess that occurs constitutively.

For example, estrogen is now being prescribed to retard bone loss. Thereis, however, some controversy over whether there is any long termbenefit to patients and whether there is any effect at all on patientsover 75 years old. Moreover, use of estrogen is believed to increase therisk of breast and endometrial cancer.

High doses of dietary calcium, with or without vitamin D has also beensuggested for postmenopausal women. However, high doses of calcium canoften have unpleasant gastrointestinal side effects, and serum andurinary calcium levels must be continuously monitored (see Khosla andRigss, Mayo Clin. Proc. 70:978–982, 1995).

Other therapeutics which have been suggested include calcitonin,bisphosphonates, anabolic steroids and sodium fluoride. Suchtherapeutics however, have undesirable side effects (e.g., calcitoninand steroids may cause nausea and provoke an immune reaction,bisphosphonates and sodium fluoride may inhibit repair of fractures,even though bone density increases modestly) that may prevent theirusage (see Khosla and Rigss, supra).

No currently practiced therapeutic strategy involves a drug thatstimulates or enhances the growth of new bone mass. The presentinvention provides compositions and methods which can be utilized toincrease bone mineralization, and thus may be utilized to treat a widevariety of conditions where it is desired to increase bone mass.Further, the present invention provides other, related advantages.

SUMMARY OF THE INVENTION

As noted above, the present invention provides a novel class or familyof TGF-beta binding-proteins, as well as assays for selecting compoundswhich increase bone mineral content and bone mineral density, compoundswhich increase bone mineral content and bone mineral density and methodsfor utilizing such compounds in the treatment or prevention of a widevariety of conditions.

Within one aspect of the present invention, isolated nucleic acidmolecules are provided, wherein said nucleic acid molecules are selectedfrom the group consisting of: (a) an isolated nucleic acid moleculecomprising sequence ID Nos. 1, 5, 7, 9, 11, 13, or, 15, or complementarysequence thereof, (b) an isolated nucleic acid molecule thatspecifically hybridizes to the nucleic acid molecule of (a) underconditions of high stringency; and (c) an isolated nucleic acid thatencodes a TGF-beta binding-protein according to (a) or (b). Withinrelated aspects of the present invention, isolated nucleic acidmolecules are provided based upon hybridization to only a portion of oneof the above-identified sequences (e.g., for (a) hybridization may be toa probe of at least 20, 25, 50, or 100 nucleotides: selected fromnucleotides 156 to 539 or 555 to 687 of Sequence ID No 1). As should bereadily evident, the necessary stringency to be utilized forhybridization may vary based upon the size of the probe. For example,for a 25-mer probe high stringency conditions could include: 60 mM TrispH 8.0, 2 mM EDTA, 5× Denhardt's, 6×SSC, 0.1% (w/v) N-laurylsarcosine,0.5% (w/v) NP40 (nonidet P-40) overnight at 45 degrees C., followed bytwo washes with with 0.2×SSC/0.1% SDS at 45–50 degrees. For a 100-merprobe under low stringency conditions, suitable conditions might includethe following: 5×SSPE, 5× Denhardt's, and 0.5% SDS overnight at 42–50degrees, followed by two washes with 2×SSPE (or 2×SSC)/0.1% SDS at 42–50degrees.

Within related aspects of the present invention, isolated nucleic acidmolecules are provided which have homology to Sequence ID Nos. 1, 5, 7,9, 11, 13, or 15, at a 50%, 60%, 75%, 80%, 90%, 95%, or 98% level ofhomology utilizing a Wilbur-Lipman algorithm. Representative examples ofsuch isolated molecules include, for example, nucleic acid moleculeswhich encode a protein comprising Sequence ID NOs. 2, 6, 10, 12, 14, or16, or have homology to these sequences at a level of 50%, 60%, 75%,80%, 90%, 95%, or 98% level of homology utilizing a Lipman-Pearsonalgorithm.

Isolated nucleic acid molecules are typically less than 100 kb in size,and within certain embodiments, less than 50 kb, 25 kb, 10 kb, or even 5kb in size. Further, isolated nucleic acid molecules, within otherembodiments, do not exist in a “library” of other unrelated nucleic acidmolecules (e.g., a subclone BAC such as described in GenBank AccessionNo. AC003098 and EMB No. AQ171546). However, isolated nucleic acidmolecules can be found in libraries of related molecules (e.g., forshuffling, such as is described in U.S. Pat. Nos. 5,837,458; 5,830,721;and 5,811,238). Finally, isolated nucleic acid molecules as describedherein do not include nucleic acid molecules which encode Dan, Cerberus,Gremlin, or SCGF (U.S. Pat. No. 5,780,263).

Also provided by the present invention are cloning vectors which containthe above-noted nucleic acid molecules, and expression vectors whichcomprise a promoter (e.g., a regulatory sequence) operably linked to oneof the above-noted nucleic acid molecules. Representative examples ofsuitable promoters include tissue-specific promoters, and viral—basedpromoters (e.g., CMV-based promoters such as CMV I-E, SV40 earlypromoter, and MuLV LTR). Expression vectors may also be based upon, orderived from viruses (e.g., a “viral vector”). Representative examplesof viral vectors include herpes simplex viral vectors, adenoviralvectors, adenovirus-associated viral vectors and retroviral vectors.Also provided are host cells containing or comprising any of above-notedvectors (including for example, host cells of human, monkey, dog, rat,or mouse origin).

Within other aspects of the present invention, methods of producingTGF-beta binding-proteins are provided, comprising the step of culturingthe aforementioned host cell containing vector under conditions and fora time sufficient to produce the TGF-beta binding protein. Withinfurther embodiments, the protein produced by this method may be furtherpurified (e.g., by column chromatography, affinity purification, and thelike). Hence, isolated proteins which are encoded by the above-notednucleic acid molecules (e.g., Sequence ID NOs. 2, 4, 6, 8, 10, 12, 14,or 16) may be readily produced given the disclosure of the subjectapplication.

It should also be noted that the aforementioned proteins, or fragmentsthereof, may be produced as fusion proteins. For example, within oneaspect fusion proteins are provided comprising a first polypeptidesegment comprising a TGF-beta binding-protein encoded by a nucleic acidmolecule as described above, or a portion thereof of at least 10, 20,30, 50, or 100 amino acids in length, and a second polypeptide segmentcomprising a non-TGF-beta binding-protein. Within certain embodiments,the second polypeptide may be a tag suitable for purification orrecognition (e.g., a polypeptide comprising multiple anionic amino acidresidues—see U.S. Pat. No. 4,851,341), a marker (e.g., green fluorescentprotein, or alkaline phosphatase), or a toxic molecule (e.g., ricin).

Within another aspect of the present invention, antibodies are providedwhich are capable of specifically binding the above-described class ofTGF-beta binding proteins (e.g., human BEER). Within variousembodiments, the antibody may be a polyclonal antibody, or a monoclonalantibody (e.g., of human or murine origin). Within further embodiments,the antibody is a fragment of an antibody which retains the bindingcharacteristics of a whole antibody (e.g., an F(ab′)₂, F(ab)₂, Fab′,Fab, or Fv fragment, or even a CDR). Also provided are hybridomas andother cells which are capable of producing or expressing theaforementioned antibodies.

Within related aspects of the invention, methods are provided detectinga TGF-beta binding protein, comprising the steps of incubating anantibody as described above under conditions and for a time sufficientto permit said antibody to bind to a TGF-beta binding protein, anddetecting the binding. Within various embodiments the antibody may bebound to a solid support to facilitate washing or separation, and/orlabeled. (e.g., with a marker selected from the group consisting ofenzymes, fluorescent proteins, and radioisotopes).

Within other aspects of the present invention, isolated oligonucleotidesare provided which hybridize to a nucleic acid molecule according toSequence ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, or 18 or the complementthereto, under conditions of high stringency. Within furtherembodiments, the oligonucleotide may be found in the sequence whichencodes Sequence ID Nos. 2, 4, 6, 8, 10, 12, 14, or 16. Within certainembodiments, the oligonucleotide is at least 15, 20, 30, 50, or 100nucleotides in length. Within further embodiments, the oligonucleotideis labeled with another molecule (e.g. an enzyme, fluorescent molecule,or radioisotope). Also provided are primers which are capable ofspecifically amplifying all or a portion of the above-mentioned nucleicacid molecules which encode TGF-beta binding-proteins. As utilizedherein, the term “specifically amplifying” should be understood to referto primers which amplify the aforementioned TGF-beta binding-proteins,and not other TGF-beta binding proteins such as Dan, Cerberus, Gremlin,or SCGF (U.S. Pat. No. 5,780,263).

Within related aspects of the present invention, methods are providedfor detecting a nucleic acid molecule which encodes a TGF-beta bindingprotein, comprising the steps of incubating an oligonucleotide asdescribed above under conditions of high stringency, and detectinghybridization of said oligonucleotide. Within certain embodiments, theoligonucleotide may be labeled and/or bound to a solid support.

Within other aspects of the present invention, ribozymes are providedwhich are capable of cleaving RNA which encodes one of theabove-mentioned TGF-beta binding-proteins (e.g., Sequence ID NOs. 2, 6,8, 10, 12, 14, or 16). Such ribozymes may be composed of DNA, RNA(including 2′-O-methyl ribonucleic acids), nucleic acid analogs (e.g.,nucleic acids having phosphorothioate linkages) or mixtures thereof.Also provided are nucleic acid molecules (e.g. DNA or cDNA) which encodethese ribozymes, and vectors which are capable of expressing orproducing the ribozymes. Representative examples of vectors includeplasmids, retrotransposons, cosmids, and viral-based vectors (e.g.,viral vectors generated at least in part from a retrovirus, adenovirus,or, adeno-associated virus). Also provided are host cells (e.g., human,dog, rat, or mouse cells) which contain these vectors. In certainembodiments, the host cell may be stably transformed with the vector.

Within further aspects of the invention, methods are provided forproducing ribozymes either synthetically, or by in vitro or in vivotranscription. Within further embodiments, the ribozymes so produced maybe further purified and/or formulated into pharmaceutical compositions(e.g., the ribozyme or nucleic acid molecule encoding the ribozyme alongwith a pharmaceutically acceptable carrier or diluent). Similarly, theantisense oligonucleotides and antibodies or other selected moleculesdescribed herein may be formulated into pharmaceutical compositions.

Within other aspects of the present invention, antisenseoligonucleotides are provided comprising a nucleic acid molecule whichhybridizes to a nucleic acid molecule according to Sequence ID NOs. 1,3, 5, 7, 9, 11, 13, or 15, or the complement thereto, and wherein saidoligonucleotide inhibits the expression of TGF-beta binding protein asdescribed herein (e.g., human BEER). Within various embodiments, theoligonucleotide is 15, 20, 25, 30, 35, 40, or 50 nucleotides in length.Preferably, the oligonucleotide is less than 100, 75, or 60 nucleotidesin length. As should be readily evident, the oligonucleotide may becomprised of one or more nucleic acid analogs, ribonucleic acids, ordeoxyribonucleic acids. Further, the oligonucleotide may be modified byone or more linkages, including for example, covalent linkage such as aphosphorothioate linkage, a phosphotriester linkage, a methylphosphonate linkage, a methylene(methylimino) linkage, a morpholinolinkage, an amide linkage, a polyamide linkage, a short chain alkylintersugar linkage, a cycloalkyl intersugar linkage, a short chainheteroatomic intersugar linkage and a heterocyclic intersugar linkage.One representative example of a chimeric oligonucleotide is provided inU.S. Pat. No. 5,989,912.

Within yet another aspect of the present invention, methods are providedfor increasing bone mineralization, comprising introducing into awarm-blooded animal an effective amount of the ribozyme as describedabove. Within related aspects, such methods comprise the step ofintroducing into a patient an effective amount of the nucleic acidmolecule or vector as described herein which is capable of producing thedesired ribozyme, under conditions favoring transcription of the nucleicacid molecule to produce the ribozyme.

Within other aspects of the invention transgenic, non-human animals areprovided. Within one embodiment a transgenic animal is provided whosegerm cells and somatic cells contain a nucleic acid molecule encoding aTGF-beta binding-protein as described above which is operably linked toa promoter effective for the expression of the gene, the gene beingintroduced into the animal, or an ancestor of the animal, at anembryonic stage, with the proviso that said animal is not a human.Within other embodiments, transgenic knockout animals are provided,comprising an animal whose germ cells and somatic cells comprise adisruption of at least one allele of an endogenous nucleic acid moleculewhich hybridizes to a nucleic acid molecule which encodes a TGF-bindingprotein as described herein, wherein the disruption preventstranscription of messenger RNA from said allele as compared to an animalwithout the disruption, with the proviso that the animal is not a human.Within various embodiments, the disruption is a nucleic acid deletion,substitution, or, insertion. Within other embodiments the transgenicanimal is a mouse, rat, sheep, pig, or dog.

Within further aspects of the invention, kits are provided for thedetection of TGF-beta binding-protein gene expression, comprising acontainer that comprises a nucleic acid molecule, wherein the nucleicacid molecule is selected from the group consisting of (a) a nucleicacid molecule comprising the nucleotide sequence of SEQ ID NOs: 1, 3, 5,7, 9, 11, 13, or 15; (b) a nucleic acid molecule comprising thecomplement of the nucleotide sequence of (a); (c) a nucleic acidmolecule that is a fragment of (a) or (b) of at least 15, 20, 30, 50,75, or, 100 nucleotides in length. Also provided are kits for thedetection of a TGF-beta binding-protein which comprise a container thatcomprise one of the TGF-beta binding protein antibodies describedherein.

For example, within one aspect of the present invention methods areprovided for determining whether a selected molecule is capable ofincreasing bone mineral content, comprising the steps of (a) mixing oneor more candidate molecules with TGF-beta-binding-protein encoded by thenucleic acid molecule according to claim 1 and a selected member of theTGF-beta family of proteins (e.g., BMP 5 or 6), (b) determining whetherthe candidate molecule alters the signaling of the TGF-beta familymember, or alters the binding of the TGF-beta binding-protein to theTGF-beta family member. Within certain embodiments, the molecule altersthe ability of TGF-beta to function as a positive regulator ofmesenchymal cell differentiation. Within this aspect of the presentinvention, the candidate molecule(s) may alter signaling or binding by,for example, either decreasing (e.g., inhibiting), or increasing (e.g.,enhancing) signaling or binding.

Within yet another aspect, methods are provided for determining whethera selected molecule is capable of increasing bone mineral content,comprising the step of determining whether a selected molecule inhibitsthe binding of TGF-beta binding-protein to bone, or an analogue thereofRepresentative examples of bone or analogues thereof includehydroxyapatite and primary human bone samples obtained via biopsy.

Within certain embodiments of the above-recited methods, the selectedmolecule is contained within a mixture of molecules and the methods mayfurther comprise the step of isolating one or more molecules which arefunctional within the assay. Within yet other embodiments, TGF-betafamily of proteins is bound to a solid support and the binding ofTGF-beta binding-protein is measured or TGF-beta binding-protein arebound to a solid support and the binding of TGF-beta proteins aremeasured.

Utilizing methods such as those described above, a wide variety ofmolecules may be assayed for their ability to increase bone mineralcontent by inhibiting the binding of the TGF-beta binding-protein to theTGF-beta family of proteins. Representative examples of such moleculesinclude proteins or peptides, organic molecules, and nucleic acidmolecules.

Within other related aspects of the invention, methods are provided forincreasing bone mineral content in a warm-blooded animal, comprising thestep of administering to a warm-blooded animal a therapeuticallyeffective amount of a molecule identified from the assays recited hereinWithin another aspect, methods are provided for increasing bone mineralcontent in a warm-blooded animal, comprising the step of administeringto a warm-blooded animal a therapeutically effective amount of amolecule which inhibits the binding of the TGF-beta binding-protein tothe TGF-beta super-family of proteins, including bone morphogenicproteins (BMPs). Representative examples of suitable molecules includeantisense molecules, ribozymes, ribozyme genes, and antibodies (e.g., ahumanized antibody) which specifically recognize and alter the activityof the TGF-beta binding-protein.

Within another aspect of the present invention, methods are provided forincreasing bone mineral content in a warm-blooded animal, comprising thesteps of (a) introducing into cells which home to the bone a vectorwhich directs the expression of a molecule which inhibits the binding ofthe TGF-beta binding-protein to the TGF-beta family of proteins and bonemorphogenic proteins (BMPs), and (b) administering the vector-containingcells to a warm-blooded animal. As utilized herein, it should beunderstood that cells “home to bone” if they localize within the bonematrix after peripheral administration. Within one embodiment, suchmethods further comprise, prior to the step of introducing, isolatingcells from the marrow of bone which home to the bone. Within a furtherembodiment, the cells which home to bone are selected from the groupconsisting of CD34+ cells and osteoblasts.

Within other aspects of the present invention, molecules are provided(preferably isolated) which inhibit the binding of the TGF-betabinding-protein to the TGF-beta super-family of proteins.

Within further embodiments, the molecules may be provided as acomposition, and can further comprise an inhibitor of bone resorption.Representative examples of such inhibitors include calcitonin, estrogen,a bisphosphonate, a growth factor having anti-resorptive activity andtamoxifen.

Representative examples of molecules which may be utilized in theafore-mentioned therapeutic contexts include, e.g., ribozymes, ribozymegenes, antisense molecules, and/or antibodies (e.g. humanizedantibodies). Such molecules may depending upon their selection, used toalter, antagonize, or agonize the signalling or binding of a TGF-betabinding-protein family member as described herein

Within various embodiments of the invention, the above-describedmolecules and methods of treatment or prevention may be utilized onconditions such as osteoporosis, osteomalasia, periodontal disease,scurvy, Cushing's Disease, bone fracture and conditions due to limbimmobilization and steroid usage.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration comparing the amino acid sequence ofHuman Dan (SEQ ID NO: 44); Human Gremlin (SEQ ID NO: 42); Human Cerberus(SEQ ID NO: 43) and Human Beer (SEQ ID NO: 45). Arrows indicate theCysteine backbone.

FIG. 2 summarizes the results obtained from surveying a variety of humantissues for the expression of a TGF-beta binding-protein gene,specifically, the Human Beer gene. A semi-quantitative ReverseTranscription-Polymerase Chain Reaction (RT-PCR) procedure was used toamplify a portion of the gene from first-strand cDNA synthesized fromtotal RNA (described in more detail in EXAMPLE 2A).

FIG. 3 summarizes the results obtained from RNA in situ hybridization ofmouse embryo sections, using a cRNA probe that is complementary to themouse Beer transcript (described in more detail in EXAMPLE 2B). Panel Ais a transverse section of 10.5 dpc embryo. Panel B is a sagittalsection of 12.5 dpc embryo and panels C and D are sagittal sections of15.5 dpc embryos.

FIG. 4 illustrates, by western blot analysis, the specificity of threedifferent polyclonal antibodies for their respective antigens (describedin more detail in EXAMPLE 4). FIG. 4A shows specific reactivity of ananti-H. Beer antibody for H Beer antigen, but not H. Dan or H. Gremlin.FIG. 4B shows reactivity of an anti-H. Gremlin antibody for H. Gremlinantigen, but not H. Beer or H. Dan. FIG. 4C shows reactivity of ananti-H. Dan antibody for H. Dan, but not H. Beer or H. Gremlin.

FIG. 5 illustrates, by western blot analysis, the selectivity of theTGF-beta binding-protein, Beer, for BMP-5 and BMP-6, but not BMP-4(described in more detail in EXAMPLE 5).

FIG. 6 demonstrates that the ionic interaction between the TGF-betabinding-protein, Beer, and BMP-5 has a dissociation constant in the15–30 nM range.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Prior to setting forth the invention in detail, it may be helpful to anunderstanding thereof to set forth definitions of certain terms and tolist and to define the abbreviations that will be used hereinafter.

“Molecule” should be understood to include proteins or peptides (e.g.,antibodies, recombinant binding partners, peptides with a desiredbinding affinity), nucleic acids (e.g., DNA, RNA, chimeric nucleic acidmolecules, and nucleic acid analogues such as PNA); and organic orinorganic compounds.

“TGF-beta” should be understood to include any known or novel member ofthe TGF-beta super-family, which also includes bone morphogenic proteins(BMPs).

“TGF-beta receptor” should be understood to refer to the receptorspecific for a particular member of the TGF-beta super-family (includingbone morphogenic proteins (BMPs)).

“TGF-beta binding-protein” should be understood to refer to a proteinwith specific binding affinity for a particular member or subset ofmembers of the TGF-beta super-family (including bone morphogenicproteins (BMPs)). Specific examples of TGF-beta binding-proteins includeproteins encoded by Sequence ID Nos. 1, 5, 7, 9, 11, 13, and 15.

Inhibiting the “binding of the TGF-beta binding-protein to the TGF-betafamily of proteins and bone morphogenic proteins (BMPs)” should beunderstood to refer to molecules which allow the activation of TGF-betaor bone morphogenic proteins (BMPs), or allow the binding of TGF-betafamily members including bone morphogenic proteins (BMPs) to theirrespective receptors, by removing or preventing TGF-beta from binding toTGF-binding-protein. Such inhibition may be accomplished, for example,by molecules which inhibit the binding of the TGF-beta binding-proteinto specific members of the TGF-beta super-family.

“Vector” refers to an assembly which is capable of directing theexpression of desired protein. The vector must include transcriptionalpromoter elements which are operably linked to the gene(s) of interestThe vector may be composed of either deoxyribonucleic acids (“DNA”),ribonucleic acids (“RNA”), or a combination of the two (e.g., a DNA-RNAchimeric). Optionally, the vector may include a polyadenylationsequence, one or more restriction sites, as well as one or moreselectable markers such as neomycin phosphotransferase or hygromycinphosphotransferase. Additionally, depending on the host cell chosen andthe vector employed, other genetic elements such as an origin ofreplication, additional nucleic acid restriction sites, enhancers,sequences conferring inducibility of transcription, and selectablemarkers, may also be incorporated into the vectors described herein.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a TGF-binding protein that has been separated fromthe genomic DNA of a eukaryotic cell is an isolated DNA molecule Anotherexample of an isolated nucleic acid molecule is a chemically-synthesizednucleic acid molecule that is not integrated in the genome of anorganism. The isolated nucleic acid molecule may be genomic DNA, cDNA,RNA, or composed at least in part of nucleic acid analogs.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Within certain embodiments, a particular protein preparation contains anisolated polypeptide if it appears nominally as a single band onSDS-PAGE gel with Coomassie Blue staining. “Isolated” when referring toorganic molecules means that the compounds are greater than 90 percentpure utilizing methods which are well known in the art (e.g., NMR,melting point).

“Sclerosteosis” Sclerosteosis is a term that was applied by Hansen(1967) (Hansen, H. G., Sklerosteose.In: Opitz, H.; Schmid, F., Handbuchder Kinderheilkunde. Berlin: Springer (pub.) 6 1967. Pp. 351–355) to adisorder similar to van Buchem hyperostosis corticalis generalisata butpossibly differing in radiologic appearance of the bone changes and inthe presence of asymmetric cutaneous syndactyly of the index and middlefingers in many cases. The jaw has an unusually square appearance inthis condition.

“Humanized antibodies” are recombinant proteins in which murinecomplementary determining regions of monoclonal antibodies have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

As used herein, an “antibody fragnent” is a portion of an antibody suchas F(ab′)₂, F(ab), Fab′, Fab, and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. For example, an anti-TGF-beta binding-proteinmonoclonal antibody fragment binds with an epitope of TGF-betabinding-protein.

The term “antibody fragment” also includes any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“sFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, enzymes, and other markermoieties.

As used herein, an “immunoconjugate” is a molecule comprising ananti-TGF-beta binding-protein antibody, or an antibody fragment, and adetectable label. An immunoconjugate has roughly the same, or onlyslightly reduced, ability to bind TGF-beta binding-protein afterconjugation as before conjugation.

Abbreviations: TGF-beta—“Transforming Growth Factor-beta”;TGF-bBP—“Transforming Growth Factor-beta binding-protein” (onerepresentative TGF-bBP is designated “H. Beer”); BMP—“bone morphogenicprotein”; PCR—“polymerase chain reaction”; RT-PCR—PCR process in whichRNA is first transcribed into DNA at the first step using reversetranscriptase (RT); cDNA—any DNA made by copying an RNA sequence intoDNA form.

As noted above, the present invention provides a novel class of TGF-betabinding-proteins, as well as methods and compositions for increasingbone mineral content in warm-blooded animals. Briefly, the presentinventions are based upon the unexpected discovery that a mutation inthe gene which encodes a novel member of the TGF-beta binding-proteinfamily results in a rare condition (sclerosteosis) characterized by bonemineral contents which are one- to four-fold higher than in normalindividuals. Thus, as discussed in more detail below this discovery hasled to the development of assays which may be utilized to selectmolecules which inhibit the binding of the TGF-beta binding-protein tothe TGF-beta family of proteins and bone morphogenic proteins (BMPs),and methods of utilizing such molecules for increasing the bone mineralcontent of warm-blooded animals (including for example, humans).

Discussion of the Disease Known as Sclerosteosis

Sclerosteosis is a term that was applied by Hansen (1967) (Hansen, H.G., Sklerosteose.In: Opitz, H.; Schmid, F., Handbuch derKinderheilkunde. Berlin: Springer (pub.) 6 1967. Pp. 351–355) to adisorder similar to van Buchem hyperostosis corticalis generalisata butpossibly differing in radiologic appearance of the bone changes and inthe presence of asymmetric cutaneous syndactyly of the index and middlefingers in many cases.

Sclerosteosis is now known to be an autosomal semi-dominant disorderwhich is characterized by widely disseminated sclerotic lesions of thebone in the adult. The condition is progressive. Sclerosteosis also hasa developmental aspect which is associated with syndactyly (two or morefingers are fused together). The Sclerosteosis Syndrome is associatedwith large stature and many affected individuals attain a height of sixfeet or more. The bone mineral content of homozygotes can be 1 to 6 foldover normal individuals and bone mineral density can be 1 to 4 foldabove normal values (erg., from unaffected siblings).

The Sclerosteosis Syndrome occurs primarily in Afrikaaners of Dutchdescent in South Africa. Approximately 1/140 individuals in theAfrikaaner population are carriers of the mutated gene (heterozygotes).The mutation shows 100% penetrance. There are anecdotal reports ofincreased of bone mineral density in heterozygotes with no associatedpathologies (syndactyly or skull overgrowth).

It appears at the present time that there is no abnormality of thepituitary-hypothalamus axis in Sclerosteosis. In particular, thereappears to be no over-production of growth hormone and cortisone. Inaddition, sex hormone levels are normal in affected individuals.However, bone turnover markers (osteoblast specific alkalinephosphatase, osteocalcin, type 1 procollagen C′ propeptide (PICP), andtotal alkaline phosphatase; (see Comier, C., Curr. Opin in Rheu. 7:243,1995) indicate that there is hyperosteoblastic activity associated withthe disease but that there is normal to slightly decreased osteoclastactivity as measured by markers of bone resorption (pyridinoline,deoxypryridinoline, N-telopeptide, urinary hydroxyproline, plasmatartrate-resistant acid phosphatases and galactosyl hydroxylysine (seeComier, supra)).

Sclerosteosis is characterized by the continual deposition of bonethroughout the skeleton during the lifetime of the affected individuals.In homozygotes the continual deposition of bone mineral leads to anovergrowth of bone in areas of the skeleton where there is an absence ofmechanoreceptors (skull, jaw, cranium). In homozygotes withSclerosteosis, the overgrowth of the bones of the skull leads to cranialcompression and eventually to death due to excessive hydrostaticpressure on the brain stem. In all other parts of the skeleton there isa generalized and diffuse sclerosis. Cortical areas of the long bonesare greatly thickened resulting in a substantial increase in bonestrength. Trabecular connections are increased in thickness which inturn increases the strength of the trabecular bone. Sclerotic bonesappear unusually opaque to x-rays.

As described in more detail in Example 1, the rare genetic mutation thatis responsible for the Sclerosteosis syndrome has been localized to theregion of human chromosome 17 that encodes a novel member of theTGF-beta binding-protein family (one representative example of which isdesignated “H. Beer”). As described in more detail below, based uponthis discovery, the mechanism of bone mineralization is more fullyunderstood, allowing the development of assays for molecules whichincrease bone mineralization, and use of such molecules to increase bonemineral content, and in the treatment or prevention of a wide number ofdiseases.

TGF-Beta Super-Family

The Transforming Growth Factor-beta (TGF-beta) super-family contains avariety of growth factors that share common sequence elements andstructural motifs (at both the secondary and tertiary levels). Thisprotein family is known to exert a wide spectrum of biological responseson a large variety of cell types. Many of them have important functionsduring the embryonal development in pattern formation and tissuespecification; in adults they are involved, e.g., in wound healing andbone repair and bone remodeling, and in the modulation of the immunesystem. In addition to the three TGF-beta's, the super-family includesthe Bone Morphogenic Proteins (BMPs), Activins, Inhibins, Growth andDifferentiation Factors (GDFs), and Glial-Derived Neurotrophic Factors(GDNFs). Primary classification is established through general sequencefeatures that bin a specific protein into a general sub-family.Additional stratification within the sub-family is possible due tostricter sequence conservation between members of the smaller group. Incertain instances, such as with BMP-5, BMP-6 and BMP-7, this can be ashigh as 75 percent amino acid homology between members of the smallergroup. This level of identity enables a single representative sequenceto illustrate the key biochemical elements of the sub-group thatseparates it from other members of the larger family.

TGF-beta signals by inducing the formation of hetero-oligomericcomplexes of type I and type II receptors. The crystal structure ofTGF-beta2 has been determined. The general fold of the TGF-beta2 monomercontains a stable, compact, cysteine knotlike structure formed by threedisulphide bridges. Dimerization, stabilized by one disulphide bridge,is antiparallel.

TGF-beta family members initiate their cellular action by binding toreceptors with intrinsic serine/threonine kinase activity. This receptorfamily consists of two subfamilies, denoted type I and type IIreceptors. Each member of the TGF-beta family binds to a characteristiccombination of type I and type II receptors, both of which are neededfor signaling. In the current model for TGF-beta receptor activation,TGF-beta first binds to the type II receptor (TbR-II), which occurs inthe cell membrane in an oligomeric form with activated kinase.Thereafter, the type I receptor (TbR-I), which can not bind ligand inthe absence of TbR-II, is recruited into-the complex. TbR-H thenphosphorylates TbR-I predominantly in a domain rich in glycine andserine residues (GS domain) in the juxtamembrane region, and therebyactivates TbR-I.

Thus far seven type I receptors and five type II receptors have beenidentified.

Bone Morphogenic Proteins (BMPS) are Key Regulatory Proteins inDetermining Bone Mineral Density in Humans

A major advance in the understanding of bone formation was theidentification of the bone morphogenic proteins (BMPs), also known asosteogenic proteins (OPs), which regulate cartilage and bonedifferentiation in vivo. BMPs/OPs induce endochondral bonedifferentiation through a cascade of events which include formation ofcartilage, hypertrophy and calcification of the cartilage, vascularinvasion, differentiation of osteoblasts, and formation of bone. Asdescribed above, the BMPs/OPs (BMP 2–14, and osteogenic protein 1 and-2, OP-1 and OP-2) are members of the TGF-beta super-family. Thestriking evolutionary conservation between members the BMP/OP sub-familysuggests that they are critical in the normal development and functionof animals. Moreover, the presence of multiple forms of BMPs/OPs raisesan important question about the biological relevance of this apparentredundancy. In addition to postfetal chondrogenesis and osteogenesis,the BMPs/OPs play multiple roles in skeletogenesis (including thedevelopment of craniofacial and dental tissues) and in embryonicdevelopment and organogenesis of parenchymatous organs, including thekidney. It is now understood that nature relies on common (and few)molecular mechanisms tailored to provide the emergence of specializedtissues and organs. The BMP/OP super-family is an elegant example ofnature parsimony in programming multiple specialized functions deployingmolecular isoforms with minor variation in amino acid motifs withinhighly conserved carboxy-terminal regions.

BMP Antagonism

The BMP and Activin sub-families are subject to significantpost-translational regulation. An intricate extracellular control systemexists, whereby a high affinity antagonist is synthesized and exported,and subsequently complexes selectively with BMPs or activins to disrupttheir biological activity (W. C. Smith (1999) TIG 15(1) 3–6). A numberof these natural antagonists have been identified, and based on sequencedivergence appear to have evolved independently due to the lack ofprimary sequence conservation. There has been no structural work to dateon this class of proteins. Studies of these antagonists has highlighteda distinct preference for interacting and neutralizing BMP-2 and BMP-4.Furthermore, the mechanism of inhibition seems to differ for thedifferent antagonists (S. Iemura et al., (1998) Proc Natl Acad Sci USA95 9337–9342).

Novel TGF-Beta Binding-Proteins

1. Background re: TGF-Beta Binding-proteins

As noted above, the present invention provides a novel class of TGF-betabinding-proteins that possess a nearly identical cysteine (disulfide)scaffold when compared to Human DAN, Human Gremlin, and Human Cerberus,and SCGF (U.S. Pat. No. 5,780,263) but almost no homology at thenucleotide level (for background information, see generally Hsu, D. R.,Economides, A. N., Wang, X., Eimon, P. M., Harland, R. M., “The XenopusDorsalizing Factor Gremlin Identifies a Novel Family of SecretedProteins that Antagonize BMP Activities,” Molecular Cell 1:673–483,1998).

One representative example of the novel class of TGF-betabinding-proteins is disclosed in Sequence ID Nos. 1, 5, 9, 11, 13, and15. Representative members of this class of binding proteins should alsobe understood to include variants of the TGF-beta binding-protein (e.g.,Sequence ID Nos. 5 and 7). As utilized herein, a “TGF-betabinding-protein variant gene” refers to nucleic acid molecules thatencode a polypeptide having an amino acid sequence that is amodification of SEQ ID Nos: 2, 10, 12, 14 or 16. Such variants includenaturally-occurring polymorphisms or allelic variants of TGF-betabinding-protein genes, as well as synthetic genes that containconservative amino acid substitutions of these amino acid sequences.Additional variant forms of a TGF-beta binding-protein gene are nucleicacid molecules that contain insertions or deletions of the nucleotidesequences described herein. TGF-beta binding-protein variant genes canbe identified by determining whether the genes hybridize with a nucleicacid molecule having the nucleotide sequence of SEQ ID Nos: 1, 5, 7, 9,11, 13, or 15 under stringent conditions. In addition, TGF-betabinding-protein variant genes should encode a protein having a cysteinebackbone.

As an alternative, TGF-beta binding-protein variant genes can beidentified by sequence comparison. As used herein, two amino acidsequences have “100% amino acid sequence identity” if the amino acidresidues of the two amino acid sequences are the same when aligned formaximal correspondence. Similarly, two nucleotide sequences have “100%nucleotide sequence identity” if the nucleotide residues of the twonucleotide sequences are the same when aligned for maximalcorrespondence. Sequence comparisons can be performed using standardsoftware programs such as those included in the LASERGENE bioinformaticscomputing suite, which is produced by DNASTAR (Madison, Wis.). Othermethods for comparing two nucleotide or amino acid sequences bydetermining optimal alignment are well-known to those of skill in theart (see, for example, Peruski and Peruski. The Internet and the NewBiology: Tools for Genomic and Molecular Research (ASM Press, Inc.1997), Wu et al. (eds.), “Information Superhighway and ComputerDatabases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123–151 (CRC Press, Inc. 1997), and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.1998)).

A variant TGF-beta binding-protein should have at least a 50% amino acidsequence identity to SEQ ID NOs: 2, 6, 10, 12, 14 or 16 and preferably,greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity.Alternatively, TGF-beta binding-protein variants can be identified byhaving at least a 70% nucleotide sequence identity to SEQ ID NOs: 1, 5,9, 11, 13 or 15. Moreover, the present invention contemplates TGF-betabinding-protein gene variants having greater than 75%, 80%, 85%, 90%, or95% identity to SEQ ID NO:1 Regardless of the particular method used toidentify a TGF-beta binding-protein variant gene or variant TGF-betabinding-protein, a variant TGF-beta binding-protein or a polypeptideencoded by a variant TGF-beta binding-protein gene can be functionallycharacterized by, for example, its ability to bind to and/or inhibit thesignaling of a selected member of the TGF-beta family of proteins, or byits ability to bind specifically to an anti-TGF-beta binding-proteinantibody.

The present invention includes functional fragments of TGF-betabinding-protein genes. Within the context of this invention, a“functional fragment” of a TGF-beta binding-protein gene refers to anucleic acid molecule that encodes a portion of a TGF-betabinding-protein polypeptide which either (1) possesses the above-notedfunction activity, or (2) specifically binds with an anti-TGF-betabinding-protein antibody. For example, a functional fragment of aTGF-beta binding-protein gene described herein comprises a portion ofthe nucleotide sequence of SEQ ID Nos: 1, 5, 9, 11, 13, or 15.

2. Isolation of the TGF-beta Binding-protein Gene

DNA molecules encoding a binding-protein gene can be obtained byscreening a human cDNA or genomic library using polynucleotide probesbased upon, for example, SEQ ID NO:1.

For example, the first step in the preparation of a cDNA library is toisolate RNA using methods well-known to those of skill in the art. Ingeneral, RNA isolation techniques must provide a method for breakingcells, a means of inhibiting RNase-directed degradation of RNA, and amethod of separating RNA from DNA, protein, and polysaccharidecontaminants. For example, total RNA can be isolated by freezing tissuein liquid nitrogen, grinding the frozen tissue with a mortar and pestleto lyse the cells, extracting the ground tissue with a solution ofphenol/chloroform to remove proteins, and separating RNA from theremaining impurities by selective precipitation with lithium chloride(see, for example, Ausubel et al. (eds.), Short Protocols in MolecularBiology, 3rd Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995)[“Ausubel (1995)”]; Wu et al., Methods in Gene Biotechnology, pages33–41 (CRC Press, Inc. 1997) [“Wu.(1997)”]).

Alternatively, total RNA can be isolated by extracting ground tissuewith guanidinium isothiocyanate, extracting with organic solvents, andseparating RNA from contaminants using differential centrifugation (see,for example, Ausubel (1995) at pages 4-1 to 4-6; Wu (1997) at pages33–41).

In order to construct a cDNA library, poly(A)⁺ RNA must be isolated froma total RNA preparation Poly(A)⁺ RNA can be isolated from total RNA byusing the standard technique of oligo(dT)-cellulose chromatography (see,for example, Ausubel (1995) at pages 4-11 to 4-12).

Double-stranded cDNA molecules are synthesized from poly(A)⁺ RNA usingtechniques well-known to those in the art (see, for example, Wu (1997)at pages 41–46). Moreover, commercially available kits can be used tosynthesize double-stranded cDNA molecules. For example, such kits areavailable from Life Technologies, Inc. (Gaithersburg, Md.), CLONTECH.Laboratories, Inc. (Palo Alto, Calif.), Promega Corporation (Madison,Wis.) and Stratagene Cloning Systems (La Jolla, Calif.).

The basic approach for obtaining TGF-beta binding-protein cDNA clonescan be modified by constructing a subtracted cDNA library which isenriched in TGF-binding-protein-specific cDNA molecules. Techniques forconstructing subtracted libraries are well-known to those of skill inthe art (see, for example, Sargent, “Isolation of DifferentiallyExpressed Genes,” in Meth. Enzymol. 152:423, 1987, and Wu et al. (eds.),“Construction and Screening of Subtracted and Complete Expression cDNALibraries,” in Methods in Gene Biotechnology, pages 29–65 (CRC Press,Inc. 1997)).

Various cloning vectors are appropriate for the construction of a cDNAlibrary. For example, a cDNA library can be prepared in a vector derivedfrom bacteriophage, such as a λgt10 vector (see, for example, Huynh etal., “Constructing and Screening cDNA Libraries in λgt10 and λgt11,” inDNA Cloning: A Practical Approach Vol. I, Glover (ed.), page 49 (IRLPress, 1985); Wu (1997) at pages 47–52).

Alternatively, double-stranded cDNA molecules can be inserted into aplasmid vector, such as a pBluescript vector (Stratagene CloningSystems; La Jolla, Calif.), a LambdaGEM-4 (Promega Corp.; Madison, Wis.)or other commercially available vectors. Suitable cloning vectors alsocan be obtained from the American Type Culture Collection (Rockville,Md.).

In order to amplify the cloned cDNA molecules, the cDNA library isinserted into a prokaryotic host, using standard techniques. Forexample, a cDNA library can be introduced into competent E. coli DH5cells, which can be obtained from Life Technologies, Inc. (Gaithersburg,Md.).

A human genomic DNA library can be prepared by means well-known in theart (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) atpages 307–327). Genomic DNA can be isolated by lysing tissue with thedetergent Sarkosyl, digesting the lysate with proteinase K, clearinginsoluble debris from the lysate by centrifugation, precipitatingnucleic acid from the lysate using isopropanol, and purifyingresuspended DNA on a cesium chloride density gradient.

DNA fragments that are suitable for the production of a genomic librarycan be obtained by the random shearing of genomic DNA or by the partialdigestion of genomic DNA with restriction endonucleases. Genomic DNAfragments can be inserted into a vector, such as a bacteriophage orcosmid vector, in accordance with conventional techniques, such as theuse of restriction enzyme digestion to provide appropriate termini, theuse of alkaline phosphatase treatment to avoid undesirable joining ofDNA molecules, and ligation with appropriate ligases. Techniques forsuch manipulation are well-known in the art (see, for example, Ausubel(1995) at pages 5-1 to 5-6; Wu (1997) at pages 307–327).

Nucleic acid molecules that encode a TGF-beta binding-protein gene canalso be obtained using the polymerase chain reaction (PCR) witholigonucleotide primers having nucleotide sequences that are based uponthe nucleotide sequences of the human TGF-beta binding-protein gene, asdescribed herein. General methods for screening libraries with PCR areprovided by, for example, Yu et al., “Use of the Polymerase ChainReaction to Screen Phage Libraries,” in Methods in Molecular Biology,Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.),pages 211–215 (Humana Press, Inc. 1993). Moreover, techniques for usingPCR to isolate related genes are described by, for example, Preston,“Use of Degenerate Oligonucleotide Primers and the Polymerase ChainReaction to Clone Gene Family Members,” in Methods in Molecular Biology,Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.),pages 317–337 (Humana Press, Inc. 1993).

Alternatively, human genomic libraries can be obtained from commercialsources such as Research Genetics (Huntsville, Ala.) and the AmericanType Culture Collection (Rockville, Md.).

A library containing cDNA or genomic clones can be screened with one ormore polynucleotide probes based upon SEQ ID NO:1, using standardmethods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).

Anti-TGF-beta binding-protein antibodies, produced as described below,can also be used to isolate DNA sequences that encode TGF-betabinding-protein genes from cDNA libraries. For example, the antibodiescan be used to screen λgt11 expression libraries, or the antibodies canbe used for immunoscreening following hybrid selection and translation(see, for example, Ausubel (1995) at pages 6-12 to 6-16; Margolis etal., “Screening λ expression libraries with antibody and proteinprobes,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover etal. (eds.), pages 1–14 (Oxford University Press 1995)).

The sequence of a TGF-beta binding-protein cDNA or TGF-betabinding-protein genomic fragment can be determined using standardmethods. Moreover, the identification of genomic fragments containing aTGF-beta binding-protein promoter or regulatory element can be achievedusing well-established techniques, such as deletion analysis (see,generally, Ausubel (1995)).

As an alternative, a TGF-beta binding-protein gene can be obtained bysynthesizing DNA molecules using mutually priming long oligonucleotidesand the nucleotide sequences described herein (see, for example, Ausubel(1995) at pages 8-8 to 8-9). Established techniques using the polymerasechain reaction provide the ability to synthesize DNA molecules at leasttwo kilobases in length (Adang et al., Plant Molec. Biol. 21:1131, 1993;Bambot et al., PCR Methods and Applications 2:266, 1993; Dillon et al.“Use of the Polymerase Chain Reaction for the Rapid Construction ofSynthetic Genes,” in Methods in Molecular Biology, Vol. 15: PCRProtocols: Current Methods and Applications, White (ed.), pages 263–268,(Humana Press, Inc. 1993); Holowachuk et al., PCR Methods Appl. 4:299,1995).

3. Production of TGF-beta Binding-protein Genes

Nucleic acid molecules encoding variant TGF-beta binding-protein genescan be obtained by screening various cDNA or genomic libraries withpolynucleotide probes having nucleotide sequences based upon SEQ ID NO:1, 5, 9, 11, 13, or 15, using procedures described above. TGF-betabinding-protein gene variants can also be constructed synthetically. Forexample, a nucleic acid molecule can be devised that encodes apolypeptide having a conservative amino acid change, compared with theamino acid sequence of SEQ ID NOs: 2, 6, 8, 10, 12, 14, or 16. That is,variants can be obtained that contain one or more amino acidsubstitutions of SEQ ID NOs: 2, 6, 8, 10, 12, 14 or 16, in which analkyl amino acid is substituted for an alkyl amino acid in a TGF-betabinding-protein amino acid sequence, an aromatic amino acid issubstituted for an aromatic amino acid in a TGF-beta binding-proteinamino acid sequence, a sulfur-containing amino acid is substituted for asulfur-containing amino acid in a TGF-beta binding-protein amino acidsequence, a hydroxy-containing amino acid is substituted for ahydroxy-containing amino acid in a TGF-beta binding-protein amino acidsequence, an acidic amino acid is substituted for an acidic amino acidin a TGF-beta binding-protein amino acid sequence, a basic amino acid issubstituted for a basic amino acid in a TGF-beta binding-protein aminoacid sequence, or a dibasic monocarboxylic amino acid is substituted fora dibasic monocarboxylic amino acid in a TGF-beta binding-protein aminoacid sequence.

Among the common amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine. In making such substitutions, itis important to, where possible, maintain the cysteine backbone outlinedin FIG. 1.

Conservative amino acid changes in a TGF-beta binding-protein gene canbe introduced by substituting nucleotides for the nucleotides recited inSEQ ID NO:1. Such “conservative amino acid” variants can be obtained,for example, by oligonucleotide-directed mutagenesis, linker-scanningmutagenesis, mutagenesis using the polymerase chain reaction, and thelike (see Ausubel (1995) at pages 8-10 to 8-22; and McPherson (ed.),Directed Mutagenesis: A Practical Approach (IRL Press 1991)). Thefunctional ability of such variants can be determined using a standardmethod, such as the assay described herein. Alternatively, a variantTGF-beta binding-protein polypeptide can be identified by the ability tospecifically bind anti-TGF-beta binding-protein antibodies.

Routine deletion analyses of nucleic acid molecules can be performed toobtain “functional fragments” of a nucleic acid molecule that encodes aTGF-beta binding-protein polypeptide. As an illustration, DNA moleculeshaving the nucleotide sequence of SEQ ID NO:1 can be digested with Bal31nuclease to obtain a series of nested deletions. The fragments are theninserted into expression vectors in proper reading frame, and theexpressed polypeptides are isolated and tested for activity, or for theability to bind anti-TGF-beta binding-protein antibodies. Onealternative to exonuclease digestion is to use oligonucleotide-directedmutagenesis to introduce deletions or stop codons to specify productionof a desired fragment Alternatively, particular fragments of a TGF-betabinding-protein gene can be synthesized using the polymerase chainreaction.

Standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet 240:113, 1993;Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65–72 (Nijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation, Vol. 1, Boynton etal., (eds.) pages 169–199 (Academic Press 1985); Coumailleau et al, J.Biol. Chem. 270:29270, 1995; Fukunaga et al., J. Biol. Chem. 270:25291,1995; Yamaguchi et al., Biochem. Pharmacol. 50:1295, 1995; and Meisel etal., Plant Molec. Biol. 30:1, 1996.

The present invention also contemplates functional fragments of aTGF-beta binding-protein gene that have conservative amino acid changes.

A TGF-beta binding-protein variant gene can be identified on the basisof structure by determining the level of identity with nucleotide andamino acid sequences of SEQ ID NOs: 1, 5, 9, 11, 13, or, 15 and 2, 6,10, 12, 14, or 16, as discussed above. An alternative approach toidentifying a variant gene on the basis of structure is to determinewhether a nucleic acid molecule encoding a potential variant TGF-betabinding-protein gene can hybridize under stringent conditions to anucleic acid molecule having the nucleotide sequence of SEQ ID Nos: 1,5, 9, 11, 13, or, 15, or a portion thereof of at least 15 or 20nucleotides in length. As an illustration of stringent hybridizationconditions, a nucleic acid molecule having a variant TGF-betabinding-protein sequence can bind with a fragment of a nucleic acidmolecule having a sequence from SEQ ID NO:1 in a buffer containing, forexample, 5×SSPE (1×SSPE=180 mM sodium chloride, 10 mM sodium phosphate,1 mM EDTA (pH 7.7), 5×Denhardt's solution (100×Denhardt's =2% (w/v)bovine serum albumin, 2% (w/v) Ficoll, 2% (w/v) polyvinylpyrrolidone)and 0.5% SDS incubated overnight at 55–60° C. Post-hybridization washesat high stringency are typically performed in 0.5×SSC (1×SSC=150 mMsodium chloride, 15 mM trisodium citrate) or in 0.5×SSPE at 55–60° C.

Regardless of the particular nucleotide sequence of a variant TGF-betabinding-protein gene, the gene encodes a polypeptide that can becharacterized by its functional activity, or by the ability to bindspecifically to an anti-TGF-beta binding-protein antibody. Morespecifically, variant TGF-beta binding-protein genes encode polypeptideswhich exhibit at least 50%, and preferably, greater than 60, 70, 80 or90%, of the activity of polypeptides encoded by the human TGF-betabinding-protein gene described herein.

4. Production of TGF-Beta Binding-Protein in Cultured Cells

To express a TGF-beta binding-protein gene, a nucleic acid moleculeencoding the polypeptide must be operably linked to regulatory sequencesthat control transcriptional expression in an expression vector and thenintroduced into a host cell. In addition to transcriptional regulatorysequences, such as promoters and enhancers, expression vectors caninclude translational regulatory sequences and a marker gene which issuitable for selection of cells that carry the expression vector.

Expression vectors that are suitable for production of a foreign proteinin eukaryotic cells typically contain (1) prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancemarker to provide for the growth and selection of the expression vectorin a bacterial host; (2) eukaryotic DNA elements that control initiationof transcription, such as a promoter, and (3) DNA elements that controlthe processing of transcripts, such as a transcriptiontermination/polyadenylation sequence.

TGF-beta binding-proteins of the present invention are preferablyexpressed in mammalian cells. Examples of mammalian host cells includeAfrican green monkey kidney cells (Vero; ATCC CRI 1587), human embryonickidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells(BHK-21; ATCC CRL 8544), canine kidney cells (MDCK; ATCC CCL 34),Chinese hamster ovary cells (CHO-K1; ATCC CCL61), rat pituitary cells(GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells(H4-II-E; ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1;ATCC CRL 1650) and murine embryonic cells (NIH-3T3, ATCC CRL 1658).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, in which the regulatorysignals are associated with a particular gene which has a high level ofexpression. Suitable transcriptional and translational regulatorysequences also can be obtained from mammalian genes, such as actin,collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene [Hamer et al., J. Molec. Appl. Genet. 1:273, 1982], the TK promoterof Herpes virus [McKnight, Cell 31:355, 1982], the SV40 early promoter[Benoist et al., Nature 290:304, 1981], the Rous sarcoma virus promoter[Gorman et al., Proc. Nat'l Acad Sci USA 79:6777, 1982], thecytomegalovirus promoter [Foecking et al., Gene 45:101, 1980], and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163–181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control TGF-beta binding-proteingene expression in mammalian cells if the prokaryotic promoter isregulated by a eukaryotic promoter (Zhou et al., Mol. Cell. Biol.10:4529, 1990; Kaufman et al., Nucl. Acids Res. 19:4485, 1991).

TGF-beta binding-protein genes may also be expressed in bacterial,yeast, insect, or plant cells. Suitable promoters that can be used toexpress TGF-beta binding-protein polypeptides in a prokaryotic host arewell-known to those of skill in the art and include promoters capable ofrecognizing the T4, T3, Sp6 and T7 polymerases, the P_(R) and P_(L)promoters of bacteriophage lambda, the trp, recA, heat shock, lacUV5,tac, lpp-lacSpr, phoA, and lacZ promoters of E coli, promoters of B.subtilis, the promoters of the bacteriophages of Bacillus, Streptomycespromoters, the int promoter of bacteriophage lambda, the bla promoter ofpBR322, and the CAT promoter of the chloramphenicol acetyl transferasegene. Prokaryotic promoters have been reviewed by Glick, J. Ind.Microbiol. 1:277, 1987, Watson et al., Molecular Biology of the Gene,4th Ed. (Benjamin Cummins 1987), and by Ausubel et al. (1995).

Preferred prokaryotic hosts include E. coli and Bacillus subtilus.Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF′, DH5IMCR, DHI10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (Ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, MI119, MI120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (Ed) (IRL Press 1985)).

Methods for expressing proteins in prokaryotic hosts are well-known tothose of skill in the art (see, for example, Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995); Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995); and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

The baculovirus system provides an efficient means to introduce clonedTGF-beta binding-protein genes into insect cells. Suitable expressionvectors are based, upon the Autographa californica multiple nuclearpolyhedrosis virus (AcMNPV), and contain well-known promoters such asDrosophila heat shock protein (hsp) 70 promoter, Autographa californicanuclear polyhedrosis virus immediate-early gene promoter (ie-1) and thedelayed early 39K promoter, baculovirus p10 promoter, and the Drosophilametallothionein promoter. Suitable insect host cells include cell linesderived from IPLB-Sf-21, a Spodoptera frugiperda pupal ovarian cellline, such as SJ9 (ATCC CRL 1711), Sf21AE, and Sf21 (InvitrogenCorporation; San Diego, Calif.), as well as Drosophila Schneider-2cells. Established techniques for producing recombinant, proteins inbaculovirus systems are provided by Bailey et al., “Manipulation ofBaculovirus Vectors,” in Methods in Molecular Biology, Volume 7: GeneTransfer and Expression Protocols, Murray (ed), pages 147–168 (TheHumana Press, Inc. 1991), by Patel et al., “The baculovirus expressionsystem,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover etal. (eds.), pages 205–244 (Oxford University Press 1995), by Ausubel(1995) at pages 16-37 to 16-57, by Richardson (ed.), BaculovirusExpression Protocols (The Humana Press, Inc. 1995), and by Lucknow,“Insect Cell Expression Technology,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), pages 183–218 (John Wiley & Sons,Inc. 1996).

Promoters for expression in yeast include promoters from GAL1(galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase),AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.Many yeast cloning vectors have been designed and are readily available.These vectors include YIp-based vectors, such as YIp5, YRp vectors, suchas YRp17, YEp vectors such as YEp13 and YCp vectors, such as YCp19. Oneskilled in the art will appreciate that there are a wide variety ofsuitable vectors for expression in yeast cells.

Expression vectors can also be introduced into plant protoplasts, intactplant tissues, or isolated plant cells. General methods of culturingplant tissues are provided, for example, by Miki et al, “Procedures forIntroducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67–88 (CRC Press,1993).

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transection, microprojectile-mediated delivery,electroporation, and the like. Preferably, the transfected cells areselected and propagated to provide recombinant host cells that comprisethe expression vector stably integrated in the host cell genome.Techniques for is introducing vectors into eukaryotic cells andtechniques for selecting such stable transformants using a dominantselectable marker are described, for example, by Ausubel (1995) and byMurray (ed.), Gene Transfer and Expression Protocols (Humana Press1991). Methods for introducing expression vectors into bacterial, yeast,insect, and plant cells are also provided by Ausubel (1995).

General methods for expressing and recovering foreign protein producedby a mammalian cell system is provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example.Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al(eds.), pages 59–92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc., 1995).

More generally, TGF-beta binding-protein can be isolated by standardtechniques, such as affinity chromatography, size exclusionchromatography, ion exchange chromatography, HPLC and the like.Additional variations in TGF-beta binding-protein isolation andpurification can be devised by those of skill in the art. For example,anti-TGF-beta binding-protein antibodies, obtained as described below,can be used to isolate large quantities of protein by immunoaffinitypurification.

5. Production of Antibodies to TGF-beta Binding-proteins

Antibodies to TGF-beta:binding-protein can be obtained, for example,using the product of an expression vector as an antigen. Particularlyuseful anti-TGF-beta binding-protein antibodies “bind specifically” withTGF-beta binding-protein of Sequence ID Nos. 2, 6, 10, 12, 14, or 16,but not to other TGF-beta binding-proteins such as Dan, Cerberus, SCGF,or Gremlin. Antibodies of the present invention (including fragments andderivatives thereof) may be a polyclonal or, especially a monoclonalantibody. The antibody may belong to any immunoglobulin class, and maybe for example an IgG, for example IgG₁, IgG₂, IgG₃, IgG₄; IgE; IgM; orIgA antibody. It may be of animal, for example mammalian origin, and maybe for example a murine, rat, human or other primate antibody. Wheredesired the antibody may be an internalising antibody.

Polyclonal antibodies to recombinant TGF-beta binding-protein can beprepared using methods well-known to those of skill in the art (see, forexample, Green et al., “Production of Polyclonal Antisera,” inImmunochemical Protocols (Manson, ed.), pages 1–5 (Humana Press 1992);Williams et al., “Expression of foreign proteins in E. coli usingplasmid vectors and purification of specific polyclonal antibodies.” inDNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),page 15 (Oxford University Press 1995)). Although polyclonal antibodiesare typically raised in animals such as rats, mice, rabbits, goats, orsheep, an anti-TGF-beta binding-protein antibody of the presentinvention may also be derived from a subhuman primate antibody. Generaltechniques for raising diagnostically and therapeutically usefulantibodies in baboons may be found, for example, in Goldenberg et al.,international patent publication No. WO 91/11465 (1991), and in Losmanet al., Int. J. Cancer 46:310. 1990.

The antibody should comprise at least a variable region domain. Thevariable region domain may be of any size or amino acid composition andwill generally comprise at least one hypervariable amino acid sequenceresponsible for antigen binding embedded in a framework sequence. Ingeneral terms the variable (V) region domain may be any suitablearrangement of immunoglobulin heavy (V_(H)) and/or light (V_(L)) chainvariable domains. Thus for example the V region domain may be monomericand be a V_(H) or V_(L) domain where these are capable of independentlybinding antigen with acceptable affinity. Alternatively the V regiondomain may be dimeric and contain V_(H)–V_(H), V_(H)–V_(L), orV_(L)–V_(L), dimers in which the V_(H) and V_(L) chains arenon-covalently associated (abbreviated hereinafter as F_(V)). Wheredesired, however, the chains may be covalently coupled either directly,for example via a disulphide bond between the two variable domains, orthrough a linker, for example a peptide linker, to form a single chaindomain (abbreviated hereinafter as scF_(V)).

The variable region domain may be any naturally occuring variable domainor an engineered version thereof By engineered version is meant avariable region domain which has been created using recombinant DNAengineering techniques. Such engineered versions include those createdfor example from natural antibody variable regions by insertions,deletions or changes in or to the amino acid sequences of the naturalantibodies. Particular examples of this type include those engineeredvariable region domains containing at least one CDR and optionally oneor more framework amino acids from one antibody and the remainder of thevariable region domain from a second antibody.

The variable region domain may be covalently attached at a C-terminalamino acid to at least one other antibody domain or a fragment thereof.Thus, for example where a V_(H) domain is present in the variable regiondomain this may be linked to an immunoglobulin C_(H)1 domain or afragment thereof. Similarly a V_(L) domain may be linked to a C_(K)domain or a fragment thereof. In this way for example the antibody maybe a Fab fragment wherein the antigen binding domain contains associatedV_(H) and V_(L) domains covalently linked at their C-termini to a CH1and C_(K) domain respectively. The CH1 domain may be extended withfurther amino acids, for example to provide a hinge region domain asfound in a Fab′ fragment, or to provide further domains, such asantibody CH2 and CH3 domains.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106, 1991;Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.). page is 166 (Cambridge University Press 1995); andWard et al. “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Antibodies for use in the invention may in general be monoclonal(prepared by conventional immunisation and cell fusion procedures) or inthe case of fragments, derived therefrom using any suitable standardchemical e.g. reduction or enzymatic cleavage and/or digestiontechniques, for example by treatment with pepsin.

More specifically, monoclonal anti-TGF-beta binding-protein antibodiescan be generated utilizing a variety of techniques. Rodent monoclonalantibodies to specific antigens may be obtained by methods known tothose skilled in the art (see, for example, Kohler et al., Nature256:495, 1975; and Coligan et al. (eds.), Current Protocols inImmunology, 1:2.5.1–2.6.7 (John Wiley & Sons 1991) [“Coligan”]; Picksleyet al., “Production of monoclonal antibodies against proteins expressedin E. coli,” in DNA Cloning 2: Expression Systems, 2nd Edition, Gloveret al. (eds), page 93 (Oxford University Press 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a TGF-beta binding-protein gene product,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to theantigen, culturing the clones that produce antibodies to the antigen,and isolating the antibodies from the hybridoma cultures.

In addition, an anti-TGF-beta binding-protein antibody of the presentinvention may be derived from a human monoclonal antibody. Humanmonoclonal antibodies are obtained from transgenic mice that have beenengineered to produce specific human antibodies in response toantigenic-challenge. In this technique, elements of the human heavy andlight chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described, forexample, by Green et al., Nature Genet. 7:13, 1994; Lonberg et al.,Nature 368:856, 1994; and Taylor et al., Int. Immun. 6:579, 1994.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1–2.7.12 and pages 2.9.1–2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79–104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-TGF-beta binding-protein antibodies. Such antibody fragments can beobtained, for example, by proteolytic hydrolysis of the antibody.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. As an illustration, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent to produce 3.5S Fab′monovalent fragments. Optionally, the cleavage reaction can be performedusing a blocking group for the sulfhydryl groups that result fromcleavage of disulfide linkages. As an alternative, an enzymatic cleavageusing pepsin produces two monovalent Fab fragments and an Fc fragmentdirectly. These methods are described, for example, by Goldenberg, U.S.Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys. 89:230,1960, Porter, Biochem. J. 73:119, 1959, Edelman et al., in Methods inEnzymology 1:422 (Academic Press 1967), and by Coligan at pages2.8.1–2.8.10 and 2.10.–2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Alternatively, the antibody may be a recombinant or engineered antibodyobtained by the use of recombinant DNA techniques involving themanipulation and re-expression of DNA encoding antibody variable and/orconstant regions. Such DNA is known and/or is readily available from DNAlibraries including for example phage-antibody libraries (see Chiswell,D J and McCafferty, J. Tibtech 10 80–84 (1992)) or where desired can besynthesised. Standard molecular biology and/or chemistry procedures maybe used to sequence and manipulate the DNA, for example, to introducecodons to create cysteine residues, to modify, add or delete other aminoacids or domains as desired.

From here, one or more replicable expression vectors containing the DNAmay be prepared and used to transform an appropriate cell line, e.g. anon-producing myeloma cell line, such as a mouse NSO line or abacterial, e.g. E. coli line, in which production of the antibody willoccur. In order to obtain efficient transcription and translation, theDNA sequence in each vector should include appropriate regulatorysequences, particularly a promoter and leader sequence operably linkedto the variable domain sequence. Particular methods for producingantibodies in this way are generally well known and routinely used. Forexample, basic molecular biology procedures are described by Maniatis etal (Molecular Cloning, Cold Spring Harbor Laboratory, New York, 1989);DNA sequencing can be performed as described in Sanger et al (PNAS 74,5463, (1977)) and the Amersham International plc sequencing handbook;and site directed mutagenesis can be carried out according to the methodof Kramer et al (Nucl. Acids Res. 12, 9441, (1984)) and the AnglianBiotechnology Ltd handbook. Additionally, there are numerouspublications, detailing techniques suitable for the preparation ofantibodies by manipulation of DNA, creation of expression vectors andtransformation of appropriate cells, for example as reviewed by MountainA and Adair, J R in Biotechnology and Genetic Engineering Reviews (ed.Tombs, M P, 10, Chapter 1, 1992, Intercept, Andover, UK) and inInternational Patent Specification No. WO 91/09967.

Where desired, the antibody according to the invention may have one ormore effector or reporter molecules attached to it and the inventionextends to such modified proteins. The effector or reporter moleculesmay be attached to the antibody through any available amino acidside-chain, terminal amino acid or, where present carbohydratefunctional group located in the antibody, always provided of course thatthis does not adversely affect the binding properties and eventualusefulness of the molecule. Particular functional groups include, forexample any free amino, imino, thiol, hydroxyl, carboxyl or aldehydegroup. Attachment of the antibody and the effector and/or reportermolecule(s) may be achieved via such groups and an appropriatefunctional group in the effector or reporter molecules. The linkage maybe direct or indirect, through spacing or bridging groups.

Effector molecules include, for example, antineoplastic agents, toxins(such as enzymatically active toxins of bacterial or plant origin andfragments thereof e.g. ricin and fragments thereof) biologically activeproteins, for example enzymes, nucleic acids and fragments thereof, e.g.DNA, RNA and fragments thereof, naturally occurring and syntheticpolymers e.g. polysaccharides and polyalkylene polymers such aspoly(ethylene glycol) and derivatives thereof, radionuclides,particularly radioiodide, and chelated metals. Suitable reporter groupsinclude chelated metals, fluorescent compounds or compounds which may bedetected by NMR or ESR spectroscopy.

Particular antineoplastic agents include cytotoxic and cytostaticagents, for example alkylating agents, such as nitrogen mustards (e.g.chlorambucil, melphalan, mechlorethamine, cyclophosphamide, or uracilmustard) and derivatives thereof, triethylenephosphoramide,triethylenethiophosphor-amide, busulphan, or cisplatin; antimetabolites,such as methotrexate, fluorouracil, floxuridine, cytarabine,mercaptopurine, thioguanine, fluoroacetic acid or fluorocitric acid,antibiotics, such as bleomycins (e.g. bleomycin sulphate), doxorubicin,daunorubicin, mitomycins (e.g. mitomycin C), actinomycins (e.g.dactinomycin) plicamycin, calichaemicin and derivatives thereof, oresperamicin and derivatives thereof, mitotic inhibitors, such asetoposide, vincristine or vinblastine and derivatives-thereof;alkaloids, such as ellipticine; polyols such as taxicin-I or taxicin-II;hormones, such as androgens (e.g. dromostanolone or testolactone),progestins (e.g. megestrol acetate or medroxyprogesterone acetate),estrogens (e.g. dimethylstilbestrol diphosphate, polyestradiol phosphateor estramustine phosphate) or antiestrogens (e.g. tamoxifen);anthraquinones, such as mitoxantrone, ureas, such as hydroxyurea;hydrazines, such as procarbazine; or imidazoles, such as dacarbazine.

Particularly useful effector groups are calichaemicin and derivativesthereof (see for example South African Patent Specifications Nos.85/8794, 88/8127 and 90/2839).

Chelated metals include chelates of di-or tripositive metals having acoordination number from 2 to 8 inclusive. Particular examples of suchmetals include technetium (Tc), rhenium (Re), cobalt (Co), copper (Cu),gold (Au), silver (Ag), lead (Pb), bismuth (Bi), indium (In), gallium(Ga), yttrium (Y), terbium (Th), gadolinium (Gd), and scandium (Sc). Ingeneral the metal is preferably a radionuclide. Particular radionuclidesinclude ^(99m)Tc; ¹⁸⁶Re, ¹⁸⁸Re, ⁵⁸Co, ⁶⁰Co, ⁶⁷Cu, ¹⁹⁵Au, ¹⁹⁹Au, ¹¹⁰Ag,²⁰³Pb, ²⁰⁶Bi, ²⁰⁷Bi, ¹¹¹n, ⁶⁷Ga, ⁶⁸Ga, ⁸⁸Y, ⁹⁰Y, ¹⁶⁰Tb, ¹⁵³Gd and ⁴⁷Sc.

The chelated metal may be for example one of the above types of metalchelated with any suitable polydentate chelating agent, for exampleacyclic or cyclic polyamines, polyethers, (e.g. crown ethers andderivatives thereof); polyamides; porphyrins; and carbocyclicderivatives.

In general, the type of chelating agent will depend on the metal in use.One particularly useful group of chelating agents in conjugatesaccording to the invention, however, are acyclic and cyclic polyamines,especially polyaminocarboxylic acids, for examplediethylenetriaminepentaacetic acid and derivatives thereof, andmacrocyclic amines, e.g. cyclic tri-aza and tetra-aza derivatives (forexample as described in International Patent Specification No. WO92/22583); and polyamides, especially desferrioxamine and derivativesthereof.

Thus for example when it is desired to use a thiol group in the antibodyas the point of attachment this may be achieved through reaction with athiol reactive group present in the effector or reporter molecule.Examples of such groups include an á-halocarboxylic acid or ester, e.g.iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone, or adisulphide. These and other suitable linking procedures are generallyand more particularly described in International Patent SpecificationsNos. WO 93/06231, WO 92/22583, WO 90/091195 and WO 89/01476.

Assays for Selecting Molecules which Increase Bone Density

As discussed above, the present invention provides methods for selectingand/or isolating compounds which are capable of increasing bone density.For example, within one aspect of the present invention methods areprovided for determining whether a selected molecule is capable ofincreasing bone mineral content, comprising the steps of (a) mixing aselected molecule with TGF-beta binding protein and a selected member ofthe TGF-beta family of proteins, (b) determining whether the selectedmolecule stimulates signaling by the TGF-beta family of proteins, orinhibits the binding of the TGF-beta binding protein to the TGF-betafamily of proteins. Within certain embodiments, the molecule enhancesthe ability of TGF-beta to function as a positive regulator ofmesenchymal cell differentiation.

Within other aspects of the invention, methods are provided fordetermining whether a selected molecule is capable of increasing bonemineral content, comprising the steps of (a) exposing a selectedmolecule to cells which express TGF-beta binding-protein and (b)determining whether the expression (or activity) of TGF-betabinding-protein from said exposed cells decreases, and therefromdetermining whether the compound is capable of increasing bone mineralcontent. Within one embodiment, the cells are selected from the groupconsisting of the spontaneously transformed or untransformed normalhuman bone from bone biopsies and rat parietal bone osteoblasts. Suchmethods may be accomplished in a wide variety of assay formatsincluding, for example, Countercurrent Immuno-Electrophoresis (CIEP),Radioimmunoassays, Radioimmunoprecipitations, Enzyme-LinkedImmuno-Sorbent Assays (ELISA), Dot Blot assays, Inhibition orCompetition assays, and sandwich assays (see U.S. Pat. Nos. 4,376,110and 4,486,530; see also Antibodies: A Laboratory Manual, supra).

Representative embodiments of such assays are provided below in Examples5 and 6. Briefly, a family member of the TGF-beta super-family or aTGF-beta binding protein is first bound to a solid phase, followed byaddition of a candidate molecule. The labeled family member of theTGF-beta super-family or a TGF-beta binding protein is then added to theassay, the solid phase washed, and the quantity of bound or labeledTGF-beta super-family member or TGF-beta binding protein on thesolid-support determined. Molecules which are suitable for use inincreasing bone mineral content as described herein are those moleculeswhich decrease the binding of TGF-beta binding protein to a member ormembers of the TGF-beta super-family in a statistically significantmanner. Obviously, assays suitable for use within the present inventionshould not be limited to the embodiments described within Examples 2 and3. In particular, numerous parameters may be altered, such as by bindingTGF-beta to a solid phase, or by elimination of a solid phase entirely.

Within other aspects of the invention, methods are provided fordetermining whether a selected molecule is capable of increasing bonemineral content, comprising the steps of (a) exposing a selectedmolecule to cells which express TGF-beta and (b) determining whether theactivity of TGF-beta from said exposed cells is altered, and therefromdetermining whether the compound is capable of increasing bone mineralcontent. Similar to the above described methods, a wide variety ofmethods may be utilized to assess the changes of TGF-betabinding-protein expression due to a selected test compound.

For example, within one aspect of the present invention methods areprovided for determining whether a selected molecule is capable ofincreasing bone mineral content, comprising the steps of (a) mixing aselected molecule with TGF-beta-binding-protein and a selected member ofthe TGF-beta family of proteins, (b) determining whether the selectedmolecule up-regulates the signaling of the TGF-beta family of proteins,or inhibits the binding of the TGF-beta binding-protein to the TGF-betafamily of proteins. Within certain embodiments, the molecule enhancesthe ability of TGF-beta to function as a positive regulator ofmechemehymal cell differentiation.

Similar to the above described methods, a wide variety of methods may beutilized to assess stimulation of TGF-beta due to a selected testcompound. One such representative method is provided below in Example 6(see also Durham et al., Endo. 136:1374–1380.

Within yet other aspects of the present invention, methods are providedfor determining whether a selected molecule is capable of increasingbone mineral content, comprising the step of determining whether aselected molecule inhibits the binding of TGF-beta binding-protein tobone, or an analogue thereof. As utilized herein, it should beunderstood that bone or analogues thereof refers to hydroxyapatite, or asurface composed of a powdered form of bone, crushed bone or intactbone. Similar to the above described methods, a wide variety of methodsmay be utilized to assess the inhibition of TGF-beta binding-proteinlocalization to bone matrix. One such representative method is providedbelow in Example 7.

It should be noted that while the methods recited herein may refer tothe analysis of an individual test molecule, that the present inventionshould not be so limited. In particular, the selected molecule may becontained within a mixture of compounds. Hence, the recited methods mayfurther comprise the step of isolating a molecule which inhibits thebinding of TGF-beta binding-protein to a TGF-beta family member.

CANDIDATE MOLECULES

A wide variety of molecules may be assayed for their ability to inhibitthe binding of TGF-beta binding-protein to a TGF-beta family member.Representative examples which are discussed in more detail below includeorganic molecules, proteins or peptides, and nucleic acid molecules.Although it should be evident from the discussion below that thecandidate molecules described herein may be utilized in the assaysdescribed herein, it should also be readily apparent that such moleculescan also be utilized in a variety of diagnostic and therapeutic settins.

1. Organic Molecules

Numerous organic molecules may be assayed for their ability to inhibitthe binding of TGF-beta binding-protein to a TGF-beta family member.

For example, within one embodiment of the invention suitable organicmolecules may be selected from either a chemical library, whereinchemicals are assayed individually, or from combinatorial chemicallibraries where multiple compounds are assayed at once, thendeconvoluted to determine and isolate the most active compounds.

Representative examples of such combinatorial chemical libraries includethose described by Agrafiotis et al., “System and method ofautomatically generating chemical compounds with desired properties,”U.S. Pat. No. 5,463,564; Armstrong, R. W., “Synthesis of combinatorialarrays of organic compounds through the use of multiple componentcombinatorial array syntheses,” WO 95/02566; Baldwin, J. J. et al.,“Sulfonamide derivatives and their use,” WO 95/24186; Baldwin, J. J. etal., “Combinatorial dihydrobenzopyran library,” WO 95130642; Brenner,S., “New kit for preparing combinatorial libraries,” WO 95/16918;Chenera, B. et al., “Preparation of library of resin-bound aromaticcarbocyclic compounds,” WO 95/16712; Ellman, J. A., “Solid phase andcombinatorial synthesis of benzodiazepine compounds on a solid support,”U.S. Pat. No. 5,288,514; Felder, E. et al., “Novel combinatorialcompound libraries,” WO 95/16209; Lerner, R. et al., “Encodedcombinatorial chemical libraries,” WO 93/20242; Pavia, M. R et al., “Amethod for preparing and selecting pharmaceutically useful non-peptidecompounds from a structurally diverse universal library,” WO 95/04277;Summerton, J. E. and D. D. Weller, “Morpholino-subunit combinatoriallibrary and method,” U.S. Pat. No. 5,506,337; Holmes, C., “Methods forthe Solid Phase Synthesis of Thiazolidinones, Metathiaza ones, andDerivatives thereof,” WO 96/00148; Phillips, G. B. and G. P. Wei,“Solid-phase Synthesis of Benzimidazoles,” Tet. Letters 37:4887–90,1996; Ruhland, B. et al., “Solid-supported Combinatorial Synthesis ofStructurally Diverse β-Lactams,” J. Amer. Chem. Soc. 111:253–4, 1996;Look, G. C. et al., “The Indentification of Cyclooxygenase-1 Inhibitorsfrom 4-Thiazolidinone Combinatorial Libraries,” Bioorg and Med Chem.Letters 6:707–12, 1996.

2. Proteins and Peptides

A wide range of proteins and peptides may likewise be utilized ascandidate molecules for inhibitors of the binding of TGF-betabinding-protein to a TGF-beta family member.

a. Combinatorial Peptide Libraries

Peptide molecules which are putative inhibitors of the binding ofTGF-beta binding-protein to a TGF-beta family member may be obtainedthrough the screening of combinatorial peptide libraries. Such librariesmay either be prepared by one of skill in the art (see e.g., U.S. Pat.Nos. 4,528,266 and 4,359,535, and Patent Cooperation Treaty PublicationNos. WO 92/15679, WO 92/15677, WO 90/07862, WO 90/02809, or purchasedfrom commercially available sources (e.g., New England Biolabs Ph.D.™Phage Display Peptide Library Kit).

b. Antibodies

Antibodies which inhibit the binding of TGF-beta binding-protein to aTGF-beta family member may readily be prepared given the disclosureprovided herein. Within the context of the present invention, antibodiesare understood to include monoclonal antibodies, polyclonal antibodies,anti-idiotypic antibodies, antibody fragments (e g., Fab, and F(ab′)₂,F_(v) variable regions, or complementarity determining regions). Asdiscussed above, antibodies are understood to be specific againstTGF-beta binding-protein, or against a specific TGF-beta family member,if they bind with a K_(a) of greater than or equal to 10⁷M, preferablygreater than or equal to 10⁸M, and do not bind to other TGF-betabinding-proteins, or, bind with a K_(a) of less than or equal to 10⁶M.Furthermore, antibodies of the present invention should block or inhibitthe binding of TGF-beta binding-protein to a TGF-beta family member.

The affinity of a monoclonal antibody or binding partner, as well asinhibition of binding can be readily determined by one of ordinary skillin the art (see Scatchard, Ann. N.Y. Acad. Sci. 51:660–672, 1949).

Briefly, polyclonal antibodies may be readily generated by one ofordinary skill in the art from a variety of warm-blooded animals such ashorses, cows, various fowl, rabbits, mice, or rats. Typically, theTGF-beta binding-protein or unique peptide thereof of 13–20 amino acids(preferably conjugated to keyhole limpet id hemocyanin by cross-linkingwith glutaraldehyde) is utilized to immunize the animal throughintraperitoneal, intramuscular, intraocular, or subcutaneous injections,along with an adjuvant such as Freund's complete or incomplete adjuvantFollowing several booster immunizations, samples of serum are collectedand tested for reactivity to the protein or peptide Particularlypreferred polyclonal antisera will give a signal on one of these assaysthat is at least three times greater than background. Once the titer ofthe animal has reached a plateau in terms of its reactivity to theprotein, larger quantities of antisera may be readily obtained either byweekly bleedings, or by exsanguinating the animal.

Monoclonal antibodies may also be readily generated using conventionaltechniques (see U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and4,411,993 which are incorporated herein by reference; see alsoMonoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, andAntibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988, which are also incorporated herein byreference).

Briefly, within one embodiment a subject animal such as a rat or mouseis immunized with TGF-beta binding-protein or portion thereof asdescribed above. The protein may be admixed with an adjuvant such asFreund's complete or incomplete adjuvant in order to increase theresultant immune response Between one and three weeks after the initialimmunization the animal may be reimmunized with another boosterimmunization, and tested for reactivity to the protein utilizing assaysdescribed above. Once the animal has reached a plateau in its reactivityto the injected protein, it is sacrificed, and organs which containlarge numbers of B cells such as the spleen and lymph nodes areharvested.

Cells which are obtained from the immunized animal may be immortalizedby infection with a virus such as the Epstein-Barr virus (EBV) (seeGlasky and Reading, Hybridoma 8(4):377–389, 1989). Alternatively, withina preferred embodiment, the harvested spleen and/or lymph node cellsuspensions are fused with a suitable myeloma cell in order to create a“hybridoma” which secretes monoclonal antibody. Suitable myeloma linesinclude, for example, NS-1 (ATCC No. TIB 18), and P3×63–Ag 8.653 (ATCCNo. CRL 1580).

Following the fusion, the cells may be placed into culture platescontaining a suitable medium, such as RPMI 1640, or DMEM (Dulbecco'sModified Eagles Medium) (JRH Biosciences, Lenexa, Kans.), as well asadditional ingredients, such as fetal bovine serum (FBS, i.e., fromHyclone, Logan, Utah or JRH Biosciences). Additionally, the mediumshould contain a reagent which selectively allows for the growth offused spleen and myeloma cells such as HAT (hypoxanthine, aminopterin,and thymidine) (Sigma Chemical Co., St. Louis, Mo.). After about sevendays, the resulting fused cells or hybridomas may be screened in orderto determine the presence of antibodies which are reactive againstTGF-beta binding-protein (depending on the antigen used), and whichblock or inhibit the binding of TGF-beta binding-protein to a TGF-betafamily member.

A wide variety of assays may be utilized to determine the presence ofantibodies which are reactive against the proteins of the presentinvention, including for example countercurrent immuno-electrophoresis,radioimmunoassays, radioimmunoprecipitations, enzyme-linkedimmuno-sorbent assays (ELISA), dot blot assays, western blots,immunoprecipitation, inhibition or competition assays, and sandwichassays (see U.S. Pat. Nos. 4,376,110 and 4,486,530; see also Antibodies:A Laboratory Manual, Harlow and Lane (eds.), Cold Spring HarborLaboratory Press, 1988). Following several clonal dilutions andreassays, a hybridoma producing antibodies reactive against the desiredprotein may be isolated.

Other techniques may, also be utilized to construct monoclonalantibodies (see William D. Huse et al., “Generation of a LargeCombinational Library of the Immunoglobulin Repertoire in Phage Lambda,”Science 246:1275–1281, December 1989; see also L. Sastry et al.,“Cloning of the Immnunological Repertoire in Escherichia coli forGeneration of Monoclonal Catalytic Antibodies: Construction of a HeavyChain Variable Region-Specific cDNA Library,” Proc. Natl. Acad. Sci. USA86:5728–5732, August 1989; see also Michelle Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas,” Strategies in Molecular Biology 3:1–9, January 1990). Thesereferences describe a commercial system available from Stratagene (LaJolla, Calif.) which enables the production of antibodies throughrecombinant techniques. Briefly, mRNA is isolated from a B cellpopulation, and utilized to create heavy and light chain immunoglobulincDNA expression libraries in the λImmunoZap(H) and λImmunoZap(L)vectors. These vectors may be screened individually or co-expressed toform Fab fragments or antibodies (see Huse et al., supra; see alsoSastry et al., supra). Positive plaques may subsequently be converted toa non-lytic plasmid which allows high level expression of monoclonalantibody fragments from E. coli.

Similarly, portions or fragments, such as Fab and Fv fragments, ofantibodies may also be constructed utilizing conventional enzymaticdigestion or recombinant DNA techniques to incorporate the variableregions of a gene which encodes a specifically binding antibody. Withinone embodiment, the genes which encode the variable region from ahybridoma producing a monoclonal antibody of interest are amplifiedusing nucleotide primers for the variable region. These primers may besynthesized by one of ordinary skill in the art, or may be purchasedfrom commercially available sources. Stratagene (La Jolla, Calif.) sellsprimers for mouse and human variable regions including, among others,primers for V_(Ha), V_(Hb), V_(Hc), V_(Hd), C_(H1), V_(L) and C_(L)regions. These primers may be utilized to amplify heavy or light chainvariable regions, which may then be inserted into vectors such asImmunoZAP™ H or ImmunoZAP™ L (Stratagene), respectively. These vectorsmay then be introduced into E. coli, yeast, or mammalian-based systemsfor expression. Utilizing these techniques, large amounts of asingle-chain protein containing a fusion of the V_(H) and V_(L) domainsmay-be produced (see Bird et al., Science 242:423–426, 1988). Inaddition, such techniques may be utilized to change a “murine” antibodyto a “human” antibody, without altering the binding specificity of theantibody.

Once suitable antibodies have been obtained, they may be isolated orpurified by many techniques well known to those of ordinary skill in theart (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), ColdSpring Harbor Laboratory Press, 1988). Suitable techniques includepeptide or protein affinity columns, HPLC or RP-HPLC, purification onprotein A or protein G columns, or any combination of these techniques;

c. Mutant TGF-Beta Binding-Proteins

As described herein and below in the Examples (e.g., Examples 8 and 9),altered versions of TGF-beta binding-protein which compete with nativeTGF-beta binding-protein's ability to block the activity of a particularTGF-beta family member should lead to increased bone density. Thus,mutants of TGF-beta binding-protein which bind to the TGF-beta familymember but do not inhibit the function of the TGF-beta family memberwould meet the criteria. The mutant versions must effectively competewith the endogenous inhibitory functions of TGF-beta binding-protein.

d. Production of Proteins

Although various genes (or portions thereof) have been provided herein,it should be understood that within the context of the presentinvention, reference to one or more of these genes includes derivativesof the genes that are substantially similar to the genes (and, whereappropriate, the proteins (including peptides and polypeptides) that areencoded by the genes and their derivatives). As used herein, anucleotide sequence is deemed to be “substantially similar” if: (a) thenucleotide sequence is derived from the coding region of theabove-described genes and includes, for example, portions of thesequence or allelic variations of the sequences discussed above, oralternatively, encodes a molecule which inhibits the binding of TGF-betabinding-protein to a member of the TGF-beta family, (b) the nucleotidesequence is capable of hybridization to nucleotide sequences of thepresent invention under moderate, high or very high stringency (seeSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, NY, 1989); or (c) the DNA sequences aredegenerate as a result of the genetic code to the DNA sequences definedin (a) or (b). Further, the nucleic acid molecule disclosed hereinincludes both complementary and non-complementary sequences, providedthe sequences otherwise meet the criteria set forth herein. Within thecontext of the present invention, high stringency means standardhybridization conditions (e.g., 5×SSPE, 0.5% SDS at 65° C., or theequivalent).

The structure of the proteins encoded by the nucleic acid moleculesdescribed herein may be predicted from the primary translation productsusing the hydrophobicity plot function of, for example, P/C Gene orIntelligenetics Suite (Intelligenetics, Mountain View, Calif.), oraccording to the methods described by Kyte and Doolittle (J. Mol. Biol.157:105–132. 1982).

Proteins of the present invention may be prepared in the form of acidicor basic salts, or in neutral form. In addition, individual amino acidresidues may be modified by oxidation or reduction. Furthermore, varioussubstitutions, deletions, or additions may be made to the amino acid ornucleic acid sequences, the net effect of which is to retain or furtherenhance or decrease the biological activity of the mutant or wild-typeprotein. Moreover, due to degeneracy in the genetic code, for example,there may be considerable variation in nucleotide sequences encoding thesame amino acid sequence.

Other derivatives of the proteins disclosed herein include conjugates ofthe proteins along with other proteins or polypeptides. This may beaccomplished, for example, by the synthesis of N-terminal or C-terminalfusion proteins which may be added to facilitate purification oridentification of proteins (see U.S. Pat. No. 4,851,341, see also, Hoppet al., Bio/Technology 6:1204, 1988.) Alternatively, fusion proteinssuch as Flag/TGF-beta binding-protein be constructed in order to assistin the identification, expression, and analysis of the protein.

Proteins of the present invention may be constructed using a widevariety of techniques described herein. Further, mutations may beintroduced at particular loci by synthesizing oligonucleotidescontaining a mutant sequence, flanked by restriction sites enablingligation to fragments of the native sequence. Following ligation, theresulting reconstructed sequence encodes a derivative having the desiredamino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific (or segmentspecific) mutagenesis procedures may be employed to provide an alteredgene having particular codons altered according to the substitution,deletion, or insertion required. Exemplary methods of making thealterations set forth above are disclosed by Walder et al. (Gene 42:133,1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January1985, 12–19); Smith et al. (Genetic Engineering, Principles and Methods,Plenum Press, 1981); and Sambrook et al. (supra). Deletion or truncationderivatives of proteins (e.g., a soluble extracellular portion) may alsobe constructed by utilizing convenient restriction endonuclease sitesadjacent to the desired deletion. Subsequent to restriction, overhangsmay be filled in, and the DNA religated. Exemplary methods of making thealterations set forth above are disclosed by Sambrook et al. (MolecularCloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor LaboratoryPress, 1989).

Mutations which are made in the nucleic acid molecules of the presentinvention preferably preserve the reading frame of the coding sequences.Furthermore, the mutations will preferably not create complementaryregions that could hybridize to produce secondary mRNA structures, suchas loops or hairpins, that would adversely affect translation of themRNA. Although a mutation site may be predetermined, it is not necessarythat the nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis may be conducted at the target codon and theexpressed mutants screened for indicative biological activity.Alternatively, mutations may be introduced at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native sequence.Following ligation, the resulting reconstructed sequence encodes aderivative having the desired amino acid insertion, substitution, ordeletion.

Nucleic acid molecules which encode proteins of the present inventionmay also be constructed utilizing techniques of PCR mutagenesis,chemical mutagenesis (Drinkwater and Klinedinst, PNAS 83:3402–3406,1986), by forced nucleotide misincorporation (e.g., Liao and Wise Gene88:107–111, 1990), or by use of randomly mutagenized oligonucleotides(Horwitz et al., Genome 3:112–117, 1989)

The present invention also provides-for the manipulation and expressionof the above described genes by culturing host cells containing a vectorcapable of expressing the above-described genes. Such vectors or vectorconstructs include either synthetic or cDNA-derived nucleic acidmolecules encoding the desired protein, which are operably linked tosuitable transcriptional or translational regulatory elements. Suitableregulatory elements may be derived from a variety of sources, includingbacterial, fungal, viral, mammalian, insect, or plant genes. Selectionof appropriate regulatory elements is dependent on the host cell chosen,and may be readily accomplished by one of ordinary skill in the art.Examples of regulatory elements include: a transcriptional promoter andenhancer or RNA polymerase binding sequence, a transcriptionalterminator, and a ribosomal binding sequence, including a translationinitiation signal.

Nucleic acid molecules that encode any of the proteins described abovemay be readily expressed by a wide variety of prokaryotic and eukaryotichost cells, including bacterial, mammalian, yeast or other fungi, viral,insect, or plant cells. Methods for transforming or transfecting suchcells to express foreign DNA are well known in the art (see, e.g.,Itakura et al., U.S. Pat. No. 4,704,362; Hinnen et al., Proc. Natl.Acad. Sci. USA 75:1929–1933, 1978; Murray et al., U.S. Pat. No.4,801,542; Upshall et al., U.S. Pat. No. 4,935,349; Hagen et al., U.S.Pat. No. 4,784,950; Axel et al., U.S. Pat. No. 4,399,216; Goeddel etal., U.S. Pat. No. 4,766,075; and Sambrook et al. Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989;for plant cells see Czako and Marton, Plant Physiol. 104:1067–1071,1994; and Paszkowski et al., Biotech. 24:387–392, 1992).

Bacterial host cells suitable for carrying out the present inventioninclude E. coli, B. subtilis, Salmonella typhimurium, and variousspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus,as well as many other bacterial species well known to one of ordinaryskill in the art. Representative examples of bacterial host cellsinclude DH5α (Stratagene, LaJolla, Calif.).

Bacterial expression vectors preferably comprise a promoter whichfunctions in the host cell, one or more selectable phenotypic markers,and a bacterial origin of replication. Representative promoters includethe β-lactamase (penicillinase) and lactose promoter system (see Changet al., Nature 275:615, 1978), the T7 RNA polymerase promoter (Studieret al., Meth Enzymol. 185:60–89, 1990), the lambda promoter (Elvin etal., Gene 87:123–126, 1990), the trp promoter (Nichols and Yanofsky,Meth. in Enzymology 101:155, 1983) and the tac promoter (Russell et al.,Gene 20:231, 1982). Representative selectable markers include variousantibiotic resistance markers such as the kanamycin or ampicillinresistance genes. Many plasmids suitable for transforming host cells arewell known in the art, including among others, pBR322 (see Bolivar etal. Gene 2:95, 1977), the pUC plasmids pUC18, pUC19, pUC118, pUC119 (seeMessing, Meth. in Enzymology 101:20–77, 1983 and Vieira and Messing,Gene 19:259–268, 1982), and pNH8A, pNH16a, pNH18a, and Bluescript M13(Stratagene, La Jolla, Calif.).

Yeast and fungi host cells suitable for carrying out the presentinvention include, among others, Saccharomyces pombe, Saccharomycescerevisiae, the genera Pichia or Kluyveromyces and various species ofthe genus Aspergillus (McKnight et al., U.S. Pat. No. 4,935,349).Suitable expression vectors for yeast and fungi include, among others,YCp50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3(Turnbull, Bio/Technology 7:169, 1989), YRp7 (Struhl et al., Proc. Natl.Acad. Sci. USA 76:1035–1039, 1978), YEp13 (Broach et al., Gene8:121–133, 1979), pJDB249 and pJDB219 (Beggs, Nature 275:104–108, 1978)and derivatives thereof.

Preferred promoters for use in yeast include promoters from yeastglycolytic genes (Hitzeman et al., J. Biol. Chem. 255:12073–12080, 1980;Alber and Kawasaki, J. Mol. Appl. Genet. 1:419–434, 1982) or alcoholdehydrogenase genes (Young et al., in Genetic Engineering ofMicroorganisms for Chemicals, Hollaender et al. (eds.), p. 355, Plenum,New York, 1982; Ammerer, Meth Enzymol. 101:192–201, 1983). Examples ofuseful promoters for fungi vectors include those derived fromAspergillus nidulans glycolytic genes, such as the adh3 promoter(McKnight et al., EMBO J. 4:2093–2099, 1985). The expression units mayalso include a transcriptional terminator. An example of a suitableterminator is the adh3 terminator (McKnight et al., ibid., 1985).

As with bacterial vectors, the yeast vectors will generally include aselectable marker, which may be one of any number of genes that exhibita dominant phenotype for which a phenotypic assay exists to enabletransformants to be selected. Preferred selectable markers are thosethat complement host cell auxotrophy, provide antibiotic resistance orenable a cell to utilize specific carbon sources, and include leu2(Broach et al., ibid), ura3 (Botstein et al., Gene 8:17, 1979), or his3(Struhl et al., ibid). Another suitable selectable marker is the catgene, which confers chloramphenicol resistance on yeast cells.

Techniques for transforming fungi are well known in the literature, andhave been described, for instance, by Beggs (ibid.), Hinnen et al.(Proc. Natl. Acad. Sci. USA 75:1929–1933, 1978), Yelton et al. (Proc.Natl. Acad. Sci. USA 81:1740–1747, 1984), and Russell (Nature301:167–169, 1983). The genotype of the host cell may contain a geneticdefect that is complemented by the selectable marker present on theexpression vector. Choice of a particular host and selectable marker iswell within the level of ordinary skill in the art.

Protocols for the transformation of yeast are also well known to thoseof ordinary skill in the art. For example, transformation may be readilyaccomplished either by preparation of spheroplasts of yeast with DNA(see Hinnen et al., PNAS USA 75:1929, 1978) or by treatment withalkaline salts such as LiCl (see Itoh et al., J. Bacteriology 153:163,1983). Transformation of fungi may also be carried out usingpolyethylene glycol as described by Cullen et al. (Bio/Technology 5:369,1987).

Viral vectors include those which comprise a promoter that directs theexpression of an isolated nucleic acid molecule that encodes a desiredprotein as described above. A wide variety of promoters may be utilizedwithin the context of the present invention, including for example,promoters such as MoMLV LTR, RSV LTR, Friend MuLV LTh, adenoviralpromoter (Ohno et al., Science 265:781–784, 1994), neomycinphosphotransferase promoter/enhancer, late parvovirus promoter (Koeringet al., Hum. Gene Therap. 5:457–463, 1994), Herpes TY promoter, SV40promoter, metallothionein IIa gene enhancer/promoter, cytomegalovirusimmediate early promoter, and the cytomegalovirus immediate latepromoter. Within particularly preferred embodiments of the invention,the promoter is a tissue-specific promoter (see e.g., WO 91/02805; EP0,415,731; and WO 90/07936). Representative examples of suitable tissuespecific promoters include neural specific enolase promoter, plateletderived growth factor beta promoter, bone morphogenic protein promoter,human alpha1-chimaerin promoter, synapsin I promoter and synapsin IIpromoter. In addition to the above-noted promoters, other viral-specificpromoters (e.g., retroviral promoters (including those noted above, aswell as others such as HIV promoters), hepatitis, herpes (e.g., EBV),and bacterial, fungal or parasitic (e.g. malarial)-specific promotersmay be utilized in order to target a specific cell or tissue which isinfected with a virus, bacteria, fungus or parasite.

Mammalian cells suitable for carrying out the present invention include,among others COS, CHO, SaOS, osteosarcomas, KS483, MG-63, primaryosteoblasts, and human or mammalian bone marrow stroma. Mammalianexpression vectors for use in carrying out the present invention willinclude a promoter capable of directing the transcription of a clonedgene or cDNA. Preferred promoters include viral promoters and cellularpromoters. Bone specific promoters include the bone sialo-protein andthe promoter for osteocalcin. Viral promoters include thecytomegalovirus immediate early promoter (Boshart et al., Cell41:521–530, 1985), cytomegalovirus immediate late promoter, SV40promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981), MMTV LTR,RSV LTR, metallothionein-1, adenovirus E1a. Cellular promoters includethe mouse metallothionein-1 promoter (Palmiter et al, U.S. Pat. No.4,579,821), a mouse V_(κ) promoter (Bergman et al., Proc. Natl. Acad.Sci. USA 81:7041–7045, 1983; Grant et al., Nucl. Acids Res. 15:5496,1987) and a mouse V_(H) promoter (Loh et al., Cell 33:85–93, 1983). Thechoice of promoter will depend, at least in part, upon the level ofexpression desire or the recipient cell line to be transfected.

Such expression vectors may also contain a set of RNA splice siteslocated downstream from the promoter and upstream from the DNA sequenceencoding the peptide or protein of interest. Preferred RNA splice sitesmay be obtained from adenovirus and/or immunoglobulin genes. Alsocontained in the expression vectors is a polyadenylation signal locateddownstream of the coding sequence of interest. Suitable polyadenylationsignals include the early or late polyadenylation signals from SV40(Kaufman and Sharp, ibid), the polyadenylation signal from theAdenovirus 5 E1B region and the human growth hormone gene terminator(DeNoto et al, Nuc. Acids Res. 9:3719–3730, 1981). The expressionvectors may include a noncoding viral leader sequence, such as theAdenovirus 2 tripartite leader, located between the promoter and the RNAsplice sites. Preferred vectors may also include enhancer sequences,such as the SV40 enhancer. Expression vectors may also include sequencesencoding the adenovirus VA RNAs. Suitable expression vectors can beobtained from commercial sources (e.g., Stratagene, La Jolla, Calif.).

Vector constructs comprising cloned DNA sequences can be introduced intocultured mammalian cells by, for example, calcium phosphate-mediatedtransfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson,Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841–845,1982), or DEAE-dextran mediated transfection (Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY,1987). To identify cells that have stably integrated the cloned DNA, aselectable marker is generally introduced into the cells along with thegene or cDNA of interest. Preferred selectable markers for use incultured mammalian cells include genes that confer resistance to drugs,such as neomycin, hygromycin, and methotrexate. The selectable markermay be an amplifiable selectable marker. Preferred amplifiableselectable markers are the DHFR gene and the neomycin resistance gene.Selectable markers are reviewed by Thilly (Mammalian Cell Technology,Butterworth Publishers, Stoneham, Mass., which is incorporated herein byreference).

Mammalian cells containing a suitable vector are allowed to grow for aperiod of time, typically 1–2 days, to begin expressing the DNAsequence(s) of interest. Drug selection is then applied to select forgrowth of cells that are expressing the selectable marker in a stablefashion. For cells that have been transfected with an amplifiable,selectable marker the drug concentration may be increased in a stepwisemanner to select for increased copy number of the cloned sequences,thereby increasing expression levels. Cells expressing the introducedsequences are selected and screened for production of the protein ofinterest in the desired form or at the desired level. Cells that satisfythese criteria can then be cloned and scaled up for production.

Protocols for the transfection of mammalian cells are well known tothose of ordinary skill in the art. Representative methods includecalcium phosphate mediated transfection, electroporation, lipofection,retroviral, adenoviral and protoplast fusion-mediated transfection (seeSambrook et al., supra). Naked vector constructs can also be taken up bymuscular cells or other suitable cells subsequent to injection into themuscle of a mammal (or other animals).

Numerous insect host cells known in the art can also be useful withinthe present invention, in light of the subject specification. Forexample, the use of baculoviruses as vectors for expressing heterologousDNA sequences in insect cells has been reviewed by Atkinson et al.(Pestic. Sci. 28:215–224,1990).

Numerous plant host cells known in the art can also be useful within thepresent invention, in light of the subject specification. For example,the use of Agrobacterium rhizogenes as vectors for expressing genes inplant cells has been reviewed by Sinkar et al. (J. Biosci. (Bangalore)11:47–58, 1987).

Within related aspects of the present invention, proteins of the presentinvention may be expressed in a transgenic animal whose germ cells andsomatic cells contain a gene which encodes the desired protein and whichis operably linked to a promoter effective for the expression of thegene. Alternatively, in a similar manner transgenic animals may beprepared that lack the desired gene (e.g., “knockout” mice). Suchtransgenics may be prepared in a variety of non-human animals, includingmice, rats, rabbits, sheep, dogs, goats and pigs (see Hammer et al.,Nature 315:680–683, 1985, Palmiter et al., Science 222:809–814, 1983,Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438–4442, 1985, Palmiterand Brinster, Cell 41:343–345, 1985, and U.S. Pat. Nos. 5,175,383,5,087,571, 4,736,866, 5,387,742, 5,347,075, 5,221,778, and 5,175,384).Briefly, an expression vector, including a nucleic acid molecule to beexpressed together with appropriately positioned expression controlsequences, is introduced into pronuclei of fertilized eggs, for example,by microinjection. Integration of the injected DNA is detected by blotanalysis of DNA from tissue samples. It is preferred that the introducedDNA be incorporated into the germ line of the animal so that it ispassed on to the animal's progeny. Tissue-specific expression may beachieved through the use of a tissue-specific promoter, or through theuse of an inducible promoter, such as the metallothionein gene promoter(Palmiter et al. 1983, ibid), which allows regulated expression of thetransgene.

Proteins can be isolated by, among other methods, culturing suitablehost and vector systems to produce the recombinant translation productsof the present invention. Supernatants from such cell lines, or proteininclusions or whole cells where the protein is not excreted into thesupernatant can then be treated by a variety of purification proceduresin order to isolate the desired proteins. For example, the supernatantmay be first concentrated using commercially available proteinconcentration filters, such as an Amicon or Millipore Pelliconultrafiltration unit. Following concentration, the concentrate may beapplied to a suitable purification matrix such as, for example, ananti-protein antibody bound to a suitable support. Alternatively, anionor cation exchange resins may be employed in order to purify theprotein. As a further alternative, one or more reverse-phase highperformance liquid chromatography (RP-HPLC) steps may be employed tofurther purify the protein. Other methods of isolating the proteins ofthe present invention are well known in the skill of the art.

A protein is deemed to be “isolated” within the context of the presentinvention if no other (undesired) protein is detected pursuant toSDS-PAGE analysis followed by Coomassie blue staining. Within otherembodiments, the desired protein can be isolated such that no other(undesired) protein is detected pursuant to SDS-PAGE analysis followedby silver staining

3. Nucleic Acid Molecules

Within other aspects of the invention, nucleic acid molecules areprovided which are capable of inhibiting TGF-beta binding-proteinbinding to a member of the TGF-beta family. For example, within oneembodiment antisense oligonucleotide molecules are provided whichspecifically inhibit expression of TGF-beta binding-protein nucleic acidsequences (see generally, Hirashima et al. in Molecular Biology of RNA:New Perspectives (M. Inouye and B. S. Dudock, eds., 1987 Academic Press,San Diego, p. 401); Oligonucleotides: Antisense Inhibitors of GeneExpression (J. S. Cohen, ed, 1989 MacMillan Press, London); Stein andCheng, Science 261:1004–1012, 1993; WO 95/10607; U.S. Pat. No.5,359,051; WO 92106693; and EP-A2-612844). Briefly, such molecules areconstructed such that they are complementary to, and able to formWatson-Crick base pairs with, a region of transcribed TGF-betabinding-protein mRNA sequence. The resultant double-stranded nucleicacid interferes with subsequent processing of the mRNA, therebypreventing protein synthesis (see Example 10).

Within other aspects of the invention, ribozymes are provided which arecapable of inhibiting the TGF-beta binding-protein binding to a memberof the TGF-beta family. As used herein, “ribozymes” are intended toinclude RNA molecules that contain anti-sense sequences for specificrecognition, and an RNA-cleaving enzymatic activity. The catalyticstrand cleaves a specific site in a target RNA at greater thanstoichiometric concentration. A wide variety of ribozymes may beutilized within the context of the present invention, including forexample, the hammerhead ribozyme (for example, as described by Forsterand Symons, Cell 48:211–220, 1987; Haseloff and Gerlach, Nature328:596–600, 1988; Walbot and Bruening, Nature 334:196, 1988; Haseloffand Gerlach, Nature 334:585, 1988); the hairpin ribozyme (for example,as described by Haseloff et al., U.S. Pat. No. 5,254,678, issued Oct.19, 1993 and Hempel et al., European Patent Publication No. 0 360 257,published Mar. 26, 1990); and Tetrahymena ribosomal RNA-based ribozymes(see Cech et al., U.S. Pat. No. 4,987,071). Ribozymes of the presentinvention typically consist of RNA, but may also be composed of DNA,nucleic acid analogs (e.g., phosphorothioates), or chimerics thereof(e.g., DNA/RNA/RNA).

4. Labels

The gene product or any of the candidate molecules described above andbelow, may be labeled with a variety of compounds, including forexample, fluorescent molecules, toxins, and radionuclides.Representative examples of fluorescent molecules include fluorescein,Phycobili proteins, such as phycoerythin rhodamine, Texas red andluciferase. Representative examples of toxins include ricin, abrindiphtheria toxin, cholera toxin, gelonin, pokeweed antiviral protein,tritin, Shigella toxin, and Pseudomonas exotoxin A. Representativeexamples of radionuclides include Cu-64, Ga-67, Ga-68, Zr-89, Ru-97,Tc-99m, Rh-105, Pd-109, In-111, I-123, I-125, I-131, Re-186, Re-188,Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212. In addition, theantibodies described above may also be labeled or conjugated to onepartner of a ligand binding pair. Representative examples includeavidin-biotin, and riboflavin-riboflavin binding protein.

Methods for conjugating or labeling the molecules described herein withthe representative labels set forth above may be readily accomplished byone of ordinary skill in the art (see Trichothecene Antibody Conjugate,U.S. Pat. No. 4,744,981; Antibody Conjugate, U.S. Pat. No. 5,106,951;Fluorogenic Materials and Labeling Techniques, U.S. Pat. No. 4,018,884;Metal Radionuclide Labeled Proteins for Diagnosis and Therapy, U.S. Pat.No. 4,897,255; and Metal Radionuclide Chelating Compounds for ImprovedChelation Kinetics, U.S. Pat. No. 4,988,496; see also Inman, Methods InEnzymology, Vol. 34, Affinity Techniques, Enzyme Purification: Part B ,Jakoby and Wilchek (eds.), Academic Press, New York, p. 30, 1974; seealso Wilchek and Bayer, “The Avidin-Biotin Complex in BioanalyticalApplications,” Anal. Biochem. 171:1–32, 1988).

Pharmaceutical Compositions

As noted above, the present invention also provides a variety ofpharmaceutical compositions, comprising one of the above-describedmolecules which inhibits the TGF-beta binding-protein binding to amember of the TGF-beta family along with a pharmaceutically orphysiologically acceptable carrier, excipients or diluents. Generally,such carriers should be nontoxic to recipients at the dosages andconcentrations employed. Ordinarily, the preparation of suchcompositions entails combining the therapeutic agent with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, by carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTAglutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents.

In addition, the pharmaceutical compositions of the present inventionmay be prepared for administration by a variety of different routes. Inaddition, pharmaceutical compositions of the present invention may beplaced within containers, along with packaging material which providesinstructions regarding the use of such pharmaceutical compositions.Generally, such instructions will include a tangible expressiondescribing the reagent concentration, as well as within certainembodiments, relative amounts of excipient ingredients or diluents(e.g., water, saline or PBS) which may be necessary to reconstitute thepharmaceutical composition.

Methods of Treatment

The present invention also provides methods for increasing the mineralcontent and mineral density of bone. Briefly, numerous conditions resultin the loss of bone mineral content, including for example, disease,genetic predisposition, accidents which result in the lack of use ofbone (e.g., due to fracture), therapeutics which effect bone resorption,or which kill bone forming cells and normal aging. Through use of themolecules described herein which inhibit the TGF-beta binding-proteinbinding to a TGF-beta family member such conditions may be treated orprevented. As utilized herein, it should be understood that bone mineralcontent has been increased, if bone mineral content has been increasedin a statistically significant manner (e.g., greater than one-halfstandard deviation), at a selected site.

A wide variety of conditions which result in loss of bone mineralcontent may be treated with the molecules described herein. Patientswith such conditions may be identified through clinical diagnosisutilizing well known techniques (see, e g., Harrison's Principles ofInternal Medicine, McGraw-Hill, Inc.). Representative examples ofdiseases that may be treated included dysplasias, wherein there isabnormal growth or development of bone. Representative examples of suchconditions include achondroplasia, cleidocrnial dysostosis,enchondromatosis, fibrous dysplasia, Gaucher's, hypophosphatemicrickets, Marfan's, multiple hereditary exotoses, neurofibromatosis,osteogenesis imperfecta, osteopetrosis, osteopoikilosis, scleroticlesions, fractures, periodontal disease, pseudoarthrosis and pyogenicosteomyelitis.

Other conditions which may be treated or prevented include a widevariety of causes of osteopenia (i.e., a condition that causes greaterthan one standard deviation of bone mineral content or density belowpeak skeletal mineral content at youth). Representative examples of suchconditions include anemic states, conditions caused steroids, conditionscaused by heparin, bone marrow disorders, scurvy, malnutrition, calciumdeficiency, idiopathic osteoporosis, congenital osteopenia orosteoporosis, alcoholism, chronic liver disease, senility,postmenopausal state, oligomenorrhea, amenorrhea, pregnancy, diabetesmellitus, hyperthyroidism, Cushing's disease, acromegaly, hypogonadism,immobilization or disuse, reflex sympathetic dystrophy syndrome,transient regional osteoporosis and osteomalacia.

Within one aspect of the present invention, bone mineral content ordensity may be increased by administering to a warm-blooded animal atherapeutically effective amount of a molecule which inhibits theTGF-beta binding-protein binding to a TGF-beta family member. Examplesof warm-blooded animals that may be treated include both vertebrates andmammals, including for example horses, cows, pigs, sheep, dogs, cats,rats and mice. Representative examples of therapeutic molecules includeribozymes, ribozyme genes, antisense oligonucleotides and antibodies(e.g., humanized antibodies).

Within other aspects of the present invention, methods are provided forincreasing bone density, comprising the step of introducing into cellswhich home to bone a vector which directs the expression of a moleculewhich inhibits the TGF-beta binding-protein binding to a member of theTGF-beta family, and administering the vector containing cells to awarm-blooded animal. Briefly, cells which home to bone may be obtaineddirectly from the bone of patients (e.g., cells obtained from the bonemarrow such as CD34+, osteoblasts, osteocytes, and the like), fromperipheral blood, or from cultures.

A vector which directs the expression of a molecule that inhibits theTGF-beta binding-protein binding to a member of the TGF-beta family isintroduced into the cells. Representative examples of suitable vectorsinclude viral vectors such as herpes viral vectors (e.g., U.S. Pat. No.5,288,641), adenoviral vectors (e.g. WO 94/26914, WO 93/9191; Kolls etal., PNAS 91(1):215–219, 1994; Kass-Eisler et al., PNAS90(24):11498–502, 1993; Guzman et al. Circulation 88(6):283848, 1993;Guzman et al., Cir. Res. 73(6):1202–1207. 1993; Zabner et al., Cell75(2):207–216, 1993; Li et al., Hum Gene Ther. 4(4):403–409, 1993;Caillaud et al., Eur. J. Neurosci. 5(10:1287–1291, 1993; Vincent et al.,Nat. Genet. 5(2):130–134, 1993; Jaffe et al., Nat. Genet. 1(5):372–378,1992; and Levrero et al., Gene 101(2):195–202, 1991), adeno-associatedviral vectors (WO 95/133365; Flotte et al., PNAS 90(22):10613–10617,1993), baculovirus vectors, parvovirus vectors (Koering et al., Hum.Gene Therap. 5:457–463, 1994), pox virus vectors (Panicali and Paoletti,PNAS 79:4927–4931, 1982; and Ozaki et al., Biochem. Biophys. Res. Comm.193(2):653–660, 1993), and retroviruses (e.g., EP 0,415,731; WO90/07936; WO 91/0285, WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat.No. 5,2i9,740; WO 93/11230; WO 93/10218). Viral vectors may likewise beconstructed which contain a mixture of different elements (e.g.,promoters, envelope sequences and the like) from different viruses, ornon-viral sources. Within various embodiments, either the viral vectoritself, or a viral particle which contains the viral vector may beutilized in the methods and compositions described below.

Within other embodiments of the invention, nucleic acid molecules whichencode a molecule which inhibits the TGF-beta binding-protein binding toa member of the TGF-beta family themselves may be administered by avariety of techniques, including, for example, administration ofasialoosomucoid (ASOR) conjugated with poly-L-lysine DNA complexes(Cristano et al., PNAS 92122–92126, 1993), DNA linked to killedadenovirus (Curiel et al., Hum. Gene Ther. 3(2):147–154, 1992),cytofectin-mediated introduction (DMRIE-DOPE, Vical, Calif.), direct DNAinjection (Acsadi et al., Nature 352:815–818,-1991); DNA ligand (Wu etal., J. of Biol. Chem. 264:16985–16987, 1989); lipofection (Felgner etal., Proc. Natl. Acad. Sci. USA 84:7413–7417, 1989); lipsomes (Pickeringet al., Circ. 89(1):13–21, 1994; and Wang et al., PNAS 84:7851–7855,1987); microprojectile bombardment (Williams et al., PNAS 88:2726–2730,1991); and direct delivery of nucleic acids which encode the proteinitself either alone (Vile and Hart, Cancer Res. 53: 3860–3864, 1993), orutilizing PEG-nucleic acid complexes.

Representative examples of molecules which may be expressed by thevectors of present invention include ribozymes and antisense molecules,each of which are discussed in more detail above.

Determination of increased bone mineral content may be determineddirectly through the use of X-rays (e.g., Dual Energy X-rayAbsorptometry or “DEXA”), or by inference through bone turnover markers(osteoblast specific alkaline phosphatase, osteocalcin, type 1procollagen C′ propeptide (PICP), and total alkaline phosphatase; seeComier, C., Curr. Opin. in Rheu. 7:243, 1995), or markers of boneresorption (pyridinoline, deoxypryridinoline, N-telopeptide, urinaryhydroxyproline, plasma tartrate-resistant acid phosphatases andgalactosyl hydroxylysine; see Comier, supra) The amount of bone mass mayalso be calculated from body weights, or utilizing other methods (seeGuinness-Hey, Metab. Bone Dis. and Rel. Res. 5:177–181, 1984).

As will be evident to one of skill in the art, the amount and frequencyof administration will depend, of course, on such factors as the natureand severity of the indication being treated, the desired response, thecondition of the patient, and so forth. Typically, the compositions maybe administered by a variety of techniques, as noted above.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Sclerosteosis Maps to the Long Arm of HumanChromosome 17

Genetic mapping of the defect responsible for sclerosteosis in humanslocalized the gene responsible for this disorder to the region of humanchromosome 17 that encodes a novel TGF-beta binding-protein familymember. In sclerosteosis, skeletal bone displays a substantial increasein mineral density relative to that of unafflicted individuals. Bone inthe head displays overgrowth as well. Sclerosteosis patients aregenerally healthy although they may exhibit variable degrees ofsyndactyly at birth and variable degrees of cranial compression andnerve compression in the skull.

Linkage analysis of the gene defect associated with sclerosteosis wasconducted by applying the homozygosity mapping method to DNA samplescollected from 24 South African Afrikaaner families in which the diseaseoccurred (Sheffield et al, 1994, Human Molecular Genetics 3:1331–1335.“Identification of a Bardet-Biedl syndrome locus on chromosome 3 andevaluation of an efficient approach to homozygosity mapping”). TheAfrikaaner population of South Africa is genetically homogeneous; thepopulation is descended from a small number of founders who colonizedthe area several centuries ago, and it has been isolated by geographicand social barriers since the founding. Sclerosteosis is rare everywherein the world outside the Afrikaaner community, which suggests that amutation in the gene was present in the founding population and hassince increased in numbers along with the increase in the population.The use of homozygosity mapping is based on the assumption that DNAmapping markers adjacent to a recessive mutation are likely to behomozygous in affected individuals from consanguineous families andisolated populations.

A set of 371 microsatellite markers (Research Genetics, Set 6) from theautosomal chromosomes was selected to type pools of DNA fromsclerosteosis patient samples. The DNA samples for this analysis camefrom 29 sclerosteosis patients in 24 families, 59 unaffected familymembers and a set of unrelated control individuals from the samepopulation. The pools consisted of 4–6 individuals, either affectedindividuals, affected individuals from consanguineous families, parentsand unaffected siblings, or unrelated controls. In the pools ofunrelated individuals and in most of the pools with affected individualsor family members analysis of the markers showed several allele sizesfor each marker. One marker, D17S1299, showed an indication ofhomozygosity: one band in several of the pools of affected individuals.

All 24 sclerosteosis families were typed with a total of 19 markers inthe region of D17S1299 (at 17q12–q21). Affected individuals from everyfamily were shown to be homozygous in this region, and 25 of the 29individuals were homozygous for a core haplotype; they each had the samealleles between D17S1787 and D17–S930. The other four individuals hadone chromosome which matched this haplotype and a second which did not.In sum, the data compellingly suggested that this 3 megabase regioncontained the sclerosteosis mutation. Sequence analysis of most of theexons in this 3 megabase region identified a nonsense mutation in thenovel TGF-beta binding-protein coding sequence (C>T mutation at position117 of Sequence ID No. 1 results in a stop codon). This mutation wasshown to be unique to sclerosteosis patients and carriers' of Afrikaanerdescen't. The identity of the gene was further confirmed by identifyinga mutation in its intron (A>T mutation at position +3 of the intron)which results in improper mRNA processing in a single, unrelated patientwith diagnosed sclerosteosis.

Example 2 Tissue-Specificity of TGF-Beta Binding-Protein Gene Expression

A. Human Beer Gene Expression by RT-PCR:

First-strand cDNA was prepared from the following total RNA samplesusing a commercially available kit (“Superscript Preamplification Systemfor First-Strand cDNA Synthesis”, Life Technologies, Rockville, Md.):human brain, human liver, human spleen, human thymus, human placenta,human skeletal muscle, human thyroid, human pituitary, human osteoblast(NHOst from Clonetics Corp., San Diego, Calif.), human osteosarcoma cellline (Saos-2, ATCC# HTB-85), human bone, human bone marrow, humancartilage, vervet monkey bone, saccharomyces cerevisiae, and humanperipheral blood monocytes. All RNA samples were purchased from acommercial source (Clontech, Palo Alto, Calif.), except the followingwhich were prepared in-house: human osteoblast, human osteosarcoma cellline, human bone, human cartilage and vervet monkey bone. These in-houseRNA samples were prepared using a commercially available kit (“TRIReagent”, Molecular Research Center, Inc., Cincinnati, Ohio).

PCR was performed on these samples, and additionally on a human genomicsample as a control. The sense Beer oligonucleotide primer had thesequence 5′-CCGGAGCTGGAGAACAACAAG-3′ (SEQ ID NO:19). The antisense Beeroligonucleotide primer had the sequence 5′-GCACTGGCCGGAGCACACC-3′ (SEQID NO:20). In addition, PCR was performed using primers for the humanbeta-actin gene, as a control. The sense beta-actin oligonucleotideprimer had the sequence 5′-AGGCCAACCGCGAGAAGATGA CC-3′ (SEQ ID NO:21).The antisense beta-actin oligonucleotide primer had the sequence5′-GAAGT CCAGGGCGACGTAGCA-3′ is (SEQ ID NO:22). PCR was performed usingstandard conditions in 25 ul reactions, with an annealing temperature of61 degrees Celsius. Thirty-two cycles of PCR were performed with theBeer primers and twenty-four cycles were performed with the beta-actinprimers.

Following amplification, 12 ul from each reaction were analyzed byagarose gel electrophoresis and ethidium bromide staining See FIG. 2A.

B. RNA In-situ Hybridization of Mouse Embryo Sections:

The full length mouse Beer cDNA (Sequence ID No. 11) was cloned into thepCR2.1 vector (Invitrogen, Carlsbad, Calif.) in the antisense and sensedirection using the manufacturer's protocol. ³⁵S-alpha-GTP-labeled cRNAsense and antisense transcripts were synthesized using in-vitrotranscription reagents supplied by Ambion, Inc (Austin, Tex.). In-situhybridization was performed according to the protocols of Lyons et al.(J. Cell Biol. 111:2427–2436. 1990).

The mouse Beer cRNA probe detected a specific message expressed in theneural tube, limb buds, blood vessels and ossifying cartilages ofdeveloping mouse embryos. Panel A in FIG. 3 shows expression in theapical ectodermal ridge (aer) of the limb (l) bud, blood vessels (bv)and the neural tube (nt). Panel B shows expression in the 4^(th)ventricle of the brain (4). Panel C shows expression in the mandible(ma) cervical vertebrae (cv), occipital bone (oc), palate (pa) and ablood vessel (bv). Panel D shows expression in the ribs (r) and a heartvalve (va). Panel A is a transverse section of 10.5 dpc embryo. Panel Bis a sagittal section of 12.5 dpc embryo and panels C and D are sagittalsections of 15.5 dpc embryos.

ba=branchial arch, h=heart, te=telencephalon (forebrain), b=brain,f=frontonasal mass, g=gut, h=heart, j=jaw, li=liver, lu—lung, ot=oticvesicle, ao=, sc=spinal cord, skm=skeletal muscle, ns=nasal sinus,th=thymus, to=tongue, fl=forelimb, di=diaphragm

Example 3 Expression and Purification of Recombinant Beer Protein

A. Expression in COS-1 Cells:

The DNA sequence encoding the full length human Beer protein wasamplified using the following PCR oligonucleotide primers: The 5′oligonucleotide primer had the sequence 5′-AAGCTTGGTACCATGCAGCTCCCAC-3′(SEQ ID NO:23) and contained a HindIII restriction enzyme site (in bold)followed by 19 nucleotides of the Beer gene starting 6 base pairs priorto the presumed amino terminal start codon (ATG). The 3′ oligonucleotideprimer had the sequence 5′-AAGCTTCTACTTGTCATCGTCGTCCTTGTAGTCGTAGGCGTTCTCCAGCT-3′ (SEQ ID NO:24) and contained a HindIIIrestriction enzyme site (in bold) followed by a reverse complement stopcodon (CTA) followed by the reverse complement of the FLAG epitope(underlined, Sigma-Aldrich Co., St Louis, Mo.) flanked by the reversecomplement of nucleotides coding for the carboxy terminal 5 amino acidsof the Beer. The PCR product was TA cloned (“Original TA Cloning Kit”,Invitrogen, Carlsbad, Calif.) and individual clones were screened by DNAsequencing. A sequence-verified clone was then digested by HindIII andpurified on a 1.5% agarose gel using a commercially available reagents(“QIAquick Gel Extraction Kit”, Qiagen Inc., Valencia, Calif.). Thisfragment was then ligated to HindIII digested, phosphatase-treatedpcDNA3.1 (Invitrogen, Carlsbad, Calif.) plasmid with T4 DNA ligase.DH10B E. coli were transformed and plated on LB, 100 μg/ml ampicillinplates. Colonies bearing the desired recombinant in the properorientation were identified by a PCR-based screen, using a 5′ primercorresponding to the T7 promoter/priming site in pcDNA3.1 and a 3′primer with the sequence 5′-GCACTGGCCGGAGCACACC-3′ (SEQ ID, NO:25) thatcorresponds to the reverse complement of internal BEER sequence. Thesequence of the cloned fragment was confirmed by DNA sequencing.

COS-1 cells (ATCC# CRL-1650) were used for transfection. 50 μg of theexpression plasmid pcDNA-Beer-Flag was transfected using a commerciallyavailable kit following protocols supplied by the manufacturer(“DEAE-Dextran Transfection Kit”, Sigma Chemical Co., St. Louis, Mo.).The final media following transfection was DMEM Wife Technologies,Rockville, Md.) containing 0.1% Fetal Bovine Serum. After 4 days inculture, the media was removed. Expression of recombinant BEER wasanalyzed by SDS-PAGE and Western Blot using anti-FLAG M2 monoclonalantibody (Sigma-Aldrich Co., St. Louis, Mo.). Purification ofrecombinant BEER protein was performed using an anti-FLAG M2 affinitycolumn (“Mammalian Transient Expression System”, Sigma-Aldrich Co., St.Louis, Mo.). The column profile was analyzed via SDS-PAGE and WesternBlot using anti-FLAG M2 monoclonal antibody.

B. Expression in SF9 Insect Cells:

The human Beer gene sequence was amplified using PCR with standardconditions and the following primers:

Sense primer: 5′-GTCGTCGGATCCATGGGGTGGCAGGCGTTCAAGAATGAT-3′ (SEQ IDNO:26) Antisense primer: 5′-GTCGTCAAGCTTCTACTTGTCATCGTCCTTGTAGTCGTA (SEQID NO:27) GGCGTTCTCCAGCTCGGC-3′

The resulting cDNA contained the coding region of Beer with twomodifications. The N-terminal secretion signal was removed and a FLAGepitope tag (Sigma) was fused in frame to the C-terminal end of theinsert. BamH1 and HindIII cloning sites were added and the gene wassubcloned into pMelBac vector (Invitrogen) for transfer into abaculoviral expression vector using standard methods.

Recombinant baculoviruses expressing Beer protein were made using theBac-N-Blue transfection kit (Invitrogen) and purified according to themanufacturers instructions.

SF9 cells (Invitrogen) were maintained in TNM_FH media (Invitrogen)containing 10% fetal calf serum. For protein expression, SF9 cultures inspinner flasks were infected at an MOI of greater than 10. Samples ofthe media and cells were taken daily for five days, and Beer expressionmonitored by western blot using an anti-FLAG M2 monoclonal antibody(Sigma) or an anti-Beer rabbit polyclonal antiserum.

After five days the baculovirus-infected SF9 cells were harvested bycentrifugation and cell associated protein was extracted from the cellpellet using a high salt extraction buffer (1.5 M NaCl, 50 mM Tris pH7.5). The extract (20 ml per 300 ml culture) was clarified bycentrifugation, dialyzed three times against four liters of Trisbuffered saline (150 mM NaCl, 50 mM Tris pH 7.5), and clarified bycentrifugation again. This high salt fraction was applied to HitrapHeparin (Pharmacia; 5 ml bed volume), washed extensively with HEPESbuffered saline (25 mM HEPES 7.5, 150 mM Nacl) and bound proteins wereeluted with a gradient from 150 mM NaCl to 1200 mM NaCl. Beer elutionwas observed at approximately 800 mM NaCl. Beer containing fractionswere supplemented to 10% glycerol and 1 mM DTT and frozen at −80 degreesC.

Example 4 Preparation and Testing of Polyclonal Antibodies to Beer,Gremlin, and Dan

A. Preparation of Antigen:

The DNA sequences of Human Beer, Human Gremlin, and Human Dan wereamplified using standard PCR methods with the following oligonucleotideprimers:

H. Beer Sense: 5′-GACTTGGATCCCAGGGGTGGCAGGCGTTC-3′ (SEQ ID NO:28)Antisense: 5′-AGCATAAGCTTCTAGTAGGCGTTCTCCAG-3′ (SEQ ID NO:29) H. GremlinSense: 5′-GACTTGGATCCGAAGGGAAAAAGAAAGGG-3′ (SEQ ID NO:30) Antisense:5′-AGCATAAGCTTTTAATCCAAATCGATGGA-3′ (SEQ ID NO:31) H. Dan Sense:5′-ACTACGAGCTCGGCCCCACCACCCATCAACAAG-3′ (SEQ ID NO:32) Antisense:5′-ACTTAGAAGCTTTCAGTCCTCAGCCCCCTCTTCC-3′ (SEQ ID NO:33)

In each case the listed primers amplified the entire coding region minusthe secretion signal sequence. These include restriction sites forsubcloning into the bacterial expression vector pQE-30 (Qiagen Inc.,Valencia, Calif.) at sites BamHI/HindIII for Beer and Gremlin, and sitesSacI/HindIII for Dan. pQE30 contains a coding sequence for a 6×His tagat the 5′ end of the cloning region. The completed constructs weretransformed into E. coli strain M-15/pRep (Qiagen Inc) and individualclones verified by sequencing. Protein expression in M-15/pRep andpurification (6×His affinity tag binding to Ni-NTA coupled to Sepharose)were performed as described by the manufacturer (Qiagen, TheQIAexpressionist).

The E. coli-derived Beer protein was recovered in significant quantityusing solubilization in 6M guanidine and dialyzed to 2–4M to preventprecipitation during storage. Gremlin and Dan protein were recovered inhigher quantity with solubilization in 6M guanidine and a postpurification guanidine concentration of 0.5M.

B. Production and Testing of Polyclonal Antibodies:

Polyclonal antibodies to each of the three antigens were produced inrabbit and in chicken hosts using standard protocols (R & R Antibody,Stanwood, Wash.; standard protocol for rabbit immunization and antiserarecovery; Short Protocols in Molecular Biology. 2nd edition. 1992.11.37–11.41. Contributors Helen M. Cooper and Yvonne Paterson; chickenantisera was generated with Strategic Biosolutions, Ramona, Calif.).

Rabbit antisera and chicken egg Igy fraction were screened for activityvia Western blot. Each of the three antigens was separated by PAGE andtransferred to 0.45 um nitrocellulose (Novex, San Diego, Calif.). Themembrane was cut into strips with each strip containing approximately 75ng of antigen. The strips were blocked in 3% Blotting Grade Block(Bio-Rad Laboratories, Hercules, Calif.) and washed 3 times in 1× Trisbuffer saline (TBS)/0.02% TWEEN buffer. The primary antibody(preimmunization bleeds, rabbit antisera or chicken egg IgY in dilutionsranging from 1:100 to 1:10,000 in blocking buffer) was incubated withthe strips for one hour with gentle rocking. A second series of threewashes 1×TBS/0.02%TWEEN was followed by an one hour incubation with thesecondary antibody (peroxidase conjugated donkey anti-rabbit, AmershamLife Science. Piscataway, N.J.; or peroxidase conjugated donkeyanti-chicken, Jackson ImmunoResearch, West Grove, Pa.). A final cycle of3× washes of 1×TBS/0.02%TWEEN was performed and the strips weredeveloped with Lumi-Light Western Blotting Substrate (Roche MolecularBiochemicals, Mannheim, Germany).

C. Antibody Cross-Reactivity Test:

Following the protocol described in the previous section, nitrocellulosestrips of Beer, Gremlin or Dan were incubated with dilutions (1:5000 and1:10,000) of their respective rabbit antisera or chicken egg IgY as wellas to antisera or chicken egg Igy (dilutions 1:1000 and 1:5000) made tothe remaining two antigens. The increased levels of nonmatchingantibodies was performed to detect low affinity binding by thoseantibodies that may be seen only at increased concentration. Theprotocol and duration of development is the same for all three bindingevents using the protocol described above. There was no antigencross-reactivity observed for any of the antigens tested.

Example 5 Interaction of Beer with TGF-Beta Super-Family Proteins

The interaction of Beer with proteins from different phylogenetic armsof the TGF-β superfamily were studied using immunoprecipitation methods.Purified TGFβ-1, TGFβ-2, TGFβ-3, BMP-4, BMP-5, BMP-6 and GDNF wereobtained from commerical sources (R&D systems; Minneapolis, Minn.). Arepresentative protocol is as follows. Partially purified Beer wasdialyzed into HEPES buffered saline (25 mM HEPES 7.5, 150 mM NaCl).Immunoprecipitations were done in 300 ul of IP buffer (150 mM NaCl, 25mM Tris pH 7.5, 1 mM EDTA, 1.4 mM β-mercaptoethanol, 0.5% triton ×100,and 10% glycerol). 30 ng recombinant human BMP-5 protein (R&D systems)was applied to 15 ul of FLAG affinity matrix (Sigma; St Louis Mo.)) inthe presence and absence of 500 ng FLAG epitope-tagged Beer. Theproteins were incubated for 4 hours@4° C. and then the affinitymatrix-associated proteins were washed 5 times in IP buffer (1 ml perwash). The bound proteins western eluted from the is affinity matrix in60 microliters of 1×SDS PAGE sample buffer. The proteins were resolvedby SDS PAGE and Beer associated BMP-5 was detected by western blot usinganti-BMP-5 antiserum (Research Diagnostics, Inc) (see FIG. 5).

BEER Ligand Binding Assay:

FLAG-Beer protein (20 ng) is added to 100 ul PBS/0.2% BSA and adsorbedinto each well of 96 well microtiter plate previously coated withanti-FLAG monoclonal antibody (Sigma; St Louis Mo.) and blocked with 10%BSA in PBS. This is conducted at room temperature for 60 minutes. Thisprotein solution is removed and the wells are washed to remove unboundprotein. BMP-5 is added to each well in concentrations ranging from 10pM to 500 nM in PBS/0.2% BSA and incubated for 2 hours at roomtemperature. The binding solution is removed and the plate washed withthree times with 200 ul volumes of PBS/0.2% BSA. BMP-5 levels are thendetected using BMP-5 anti-serum via ELISA (F. M. Ausubel et al (1998)Current Protocols in Mol. Biol. Vol 211.2.1–112.22). Specific binding iscalculated by subtracting non-specific binding from total binding andanalyzed by the LIGAND program (Munson and Podbard, Anal. Biochem., 107,p220–239, (1980).

In a variation of this method, Beer is engineered and expressed as ahuman Fc fusion protein. Likewise the ligand BMP is engineered andexpressed as mouse Fc fusion. These proteins are incubated together andthe assay conducted as described by Mellor et al using homogeneous timeresolved fluorescence detection (G. W. Mellor et al., J of BiomolScreening, 3(2) 91–99, 1998).

Example 6 Screening Assay for Inhibition of TGF-Bera Binding-ProteinBinding to TGF-Beta Family Members

The assay described above is replicated with two exceptions. First, BMPconcentration is held fixed at the Kd determined previously. Second, acollection of antagonist candidates is added at a fixed concentration(20 uM in the case of the small organic molecule collections and 1 uM inantibody studies). These candidate molecules (antagonists) of TGF-betabinding-protein binding include organic compounds derived fromcommercial or internal collections representing diverse chemicalstructures. These compounds are prepared as stock solutions in DMSO andare added to assay wells at ≦1% of final volume under the standard assayconditions. These are incubated for 2 hours at room temperature with theBMP and Beer, the solution removed and the bound BMP is quantitated asdescribed. Agents that inhibit 40% of the BMP binding observed in theabsence of compound or antibody are considered antagonists of thisinteraction. These are further evaluated as potential inhibitors basedon titration studies to determine their inhibition constants and theirinfluence on TGF-beta binding-protein binding affinity. Comparablespecificity control assays may also be conducted to establish theselectivity profile for the identified antagonist through studies usingassays dependent on the BMP ligand action (e.g. BMP/BMP receptorcompetition study).

EXAMPLE 7 Inhibition of TGF-Beta Binding-Protein Localization to BoneMatrix

Evaluation of inhibition of localization to bone matrix (hydroxyapatite)is conducted using modifications to the method of Nicolas (Nicolas, V.Calcif Tissue Int 57:206, 1995). Briefly, ¹²⁵I-labelled TGF-betabinding-protein is prepared as described by Nicolas (supra).Hydroxyapatite is added to each well of a 96 well microtiter plateequipped with a polypropylene filtration membrane (Polyfiltroninc;Weymouth Mass.) TGF-beta binding-protein is added to 0.2% albumin in PBSbuffer. The wells containing matrix are washed 3 times with this buffer.Adsorbed TGF-beta binding-protein is eluted using 0.3M NaOH andquantitated.

Inhibitor identification is conducted via incubation of TGF-betabinding-protein with test molecules and applying the mixture to thematrix as described above. The matrix is washed 3 times with 0.2%albumin in PBS buffer. Adsorbed TGF-beta binding-protein is eluted using0.3 M NaOH and quantitated. Agents that inhibit 40% of the TGF-betabinding-protein binding observed in the absence of compound or antibodyare considered bone localization inhibitors. These inhibitors arefurther characterized through dose response studies to determine theirinhibition constants and their influence on TGF-beta binding-proteinbinding affinity.

EXAMPLE 8 Construction of TGF-Beta Binding-Protein Mutant

A. Mutagenesis:

A full-length TGF-beta binding-protein cDNA in pBluescript SK serves asa template for mutagenesis. Briefly, appropriate primers (see thediscussion provided above) are utilized to generate the DNA fragment bypolymerase chain reaction using Vent DNA polymerase (New EnglandBiolabs, Beverly, Mass.). The polymerase chain reaction is run for 23cycles in buffers provided by the manufacturer using a 57° C. annealingtemperature. The product is then exposed to two restriction enzymes andafter isolation using agarose gel electrophoresis, ligated back intopRBP4-503 from which the matching sequence has been removed by enzymaticdigestion. Integrity of the mutant is verified by DNA sequencing.

B. Mammalian Cell Expression and Isolation of Mutant TGF-betabinding-Protein:

The mutant TGF-beta binding-protein cDNAs are transferred into thepcDNA3.1 mammalian expression vector described in EXAMPLE 3. Afterverifying the sequence, the resultant constructs are transfected intoCOS-1 cells, and secreted protein is purified as described in EXAMPLE 3.

Example 9 Animal Models-I Generation of Transgenic Mice Overexpressingthe Beer Gene

The ˜200 kilobase (kb) BAC clone 15G5, isolated from the CITB mousegenomic DNA library (distributed by Research Genetics, Huntsville, Ala.)was used to determine the complete sequence of the mouse Beer gene andits 5′ and 3′ flanking regions. A 41 kb SalI fragment, containing theentire gene body, plus ˜17 kb of 5′ flanking and ˜20 kb of 3′ flankingsequence was subcloned into the BamHI site of the SuperCosI cosmidvector (Stratagene, La Jolla, Calif.) and propagated in the E. colistrain DH10B. From this cosmid construct, a 35 kb MluI—AviII restrictionfragment (Sequence No. 6), including the entire mouse Beer gene, as wellas 17 kb and 14 kb of 5′ and 3′ flanking sequence, respectively, wasthen gel purified, using conventional means, and used for microinjectionof mouse zygotes (DNX Transgenics; U.S. Pat. No. 4,873,191). Founderanimals in which the cloned DNA fragment was integrated randomly intothe genome were obtained at a frequency of 5–30% of live-born pups. Thepresence of the transgene was ascertained by performing Southern blotanalysis of genomic DNA extracted from a small amount of mouse tissue,such as the tip of a tail. DNA was extracted using the followingprotocol: tissue was digested overnight at 55° C. in a lysis buffercontaining 200 mM NaCl. 100 mM Tris pH 8.5, 5 mM EDTA, 0.2% SDS and 0.5mg/ml Proteinase K. The following day, the DNA was extracted once withphenol/chloroform (50:50), once with chloroform/isoamylalcohol (24:1)and precipitated with ethanol. Upon resuspension in TE (10 mM Tris pH7.5, 1 mM EDTA) 8–10 ug of each DNA sample were digested with arestriction endonuclease, such as EcoRI, subjected to gelelectrophoresis and transferred to a charged nylon membrane, such asHyBondN+ (Amersham, Arlington Heights, Ill.). The resulting filter wasthen hybridized with a radioactively labelled fragment of DNA derivingfrom the mouse Beer gene locus, and able to recognize both a fragmentfrom the endogenous gene locus and a fragment of a different sizederiving from the transgene. Founder animals were bred to normalnon-transgenic mice to generate sufficient numbers of transgenic andnon-transgenic progeny in which to determine the effects of Beer geneoverexpression. For these studies, animals at various ages (for example,1 day, 3 weeks, 6 weeks, 4 months) are subjected to a number ofdifferent assays designed to ascertain gross skeletal formation, bonemineral density, bone mineral content, osteoclast and osteoblastactivity, extent of endochondral ossification, cartilage formation, etc.The transcriptional activity from the transgene may be determined byextracting RNA from various tissues, and using an RT-PCR assay whichtakes advantage of single nucleotide polymorphisms between the mousestrain from which the transgene is derived (129Sv/J) and the strain ofmice used for DNA microinjection [(C57BL5/J×SJL/J)F2].

Animal Models-II Disruption of the Mouse Beer Gene by HomologousRecombination

Homologous recombination in embryonic stem (ES) cells can be used toinactivate the endogenous mouse Beer gene and subsequently generateanimals carrying the loss-of-function mutation. A reporter gene, such asthe E. coli β-galactosidase gene, was engineered into the targetingvector so that its expression is controlled by the endogenous Beergene's promoter and translational initiation signal. In this way, thespatial and temporal patterns of Beer gene expression can be determinedin animals carrying a targeted allele.

The targeting vector was constructed by first cloning thedrug-selectable phosphoglycerate kinase (PGK) promoter drivenneomycin-resistance gene (neo) cassette from pGT-N29 (New EnglandBiolabs, Beverly, Mass.) into the cloning vector pSP72 (Promega,-Madson,Wis.). PCR was used to flank the PGKneo cassette with bacteriophage P1loxp sites, which are recognition sites for the P1 Cre recombinase(Hoess et al., PNAS USA, 79:3398, 1982). This allows subsequent removalof the neo-resistance marker in targeted ES cells or ES cell-derivedanimals (U.S. Pat. No. 4,959,317). The PCR primers were comprised of the34 nucleotide (ntd) loxP sequence, 15–25 ntd complementary to the 5′ and3′ ends of the PGKneo cassette, as well as restriction enzymerecognition sites (BqamHI in the sense primer and EcoRI in theanti-sense primer) for cloning into pSP72. The sequence of the senseprimer was 5′-AATCTGGATCCATAACTTCGTATAGCATACATTATACGAAGTTATCTGCAGGATTCGAGGGCCCCT-3′ (SEQ ID NO:34); sequence of the anti-sense primer was5′-AATCTGAATTCCACCGGTGTTAATTAAATAACTTCGTATAATGTATGCTATACGAAGTTATAGATCTAGAG TCAGCTTCTGA-3′ (SEQ ID NO:35).

The next step was to clone a 3.6 kb XhoI-HindIII fragment, containingthe E. coli β-galactosidase gene and SV40 polyadenylation signal frompSVβ (Clontech, Palo Alto, Calif.) into the pSP72-PGKneo plasmid. The“short arm” of homology from the mouse Beer gene locus was generated byamplifying a 2.4 kb fragment from the BAC clone 15G5. The 3′ end of thefragment coincided with the translational initiation site of the Beergene, and the anti-sense primer used in the PCR also included 30 ntdcomplementary to the 5′ end of the β-galaclosidase gene so that itscoding region could be fused to the Beer initiation site in-frame. Theapproach taken for introducing the “short arm” into thepSP72-⊖gal-PGKneo plasmid was to linearize the plasmid at a siteupstream of the β-gal gene and then to co-transform this fragment withthe “short arm” PCR product and to select for plasmids in which the PCRproduct was integrated by homologous recombination. The sense primer forthe “short arm” amplification included 30 ntd complementary to the pSP72vector to allow for this recombination event. The sequence of the senseprimer was 5′-ATTTAGGTGACACTATAGAACTCGAGCAGCTGAAGCTTAACCACATGGTGGCTCACAACCAT-3′ (SEQ ID NO:36) andthe sequence of the anti-sense primer was 5′-AACGACGGCCAGTGAATCCGTAATCATGGTCATGCTGCCAGGTGGAGGAGGGCA-3′ (SEQ ID NO:37).

The “long arm” from the Beer gene locus was generated by amplifying a6.1 kb fragment from BAC clone 15G5 with primers which also introducethe rare-cutting restriction enzyme sites SgrAI, FseI, AscI and PacI.Specifically, the sequence of the sense primer was5′-ATTACCACCGGTGACACCCGCTTCCTGACAG-3′ (SEQ ID NO:38); the sequence ofthe anti-sense primer was 5′-ATTACTTAATTAAACATGGCGCGCCATATGGCCGGCCCCTAATTGCGGCGCATCGTTAATT-3′ (SEQ ID NO:39). The resulting PCRproduct was cloned into the TA vector (Invitrogen, Carlsbad, Calif.) asan intermediate step.

The mouse Beer gene targeting construct also included a secondselectable marker, the herpes simplex virus I thymidine kinase gene(HSVTK) under the control of rous sarcoma virus long terminal repeatelement (RSV LTR). Expression of this gene renders mammalian cellssensitive (and inviable) to gancyclovir; it is therefore a convenientway to select against neomycin-resistant cells in which the constructhas integrated by a non-homologous event (U.S. Pat. No. 5,464,764). TheRSVLTR-HSVTK cassette was amplified from pPS1337 using primers thatallow subsequent cloning into the FseI and AscI sites of the “longarm”-TA vector plasmid. For this PCR, the sequence of the sense primerwas 5′-ATTACGGCCGGCCGCAAAGGAATTCAAGA TCTGA-3′ (SEQ ID NO:40); thesequence of the anti-sense primer was 5′-ATTACGGCGCGCCCCTCACAGGCCGCACCCAGCT-3′ (SEQ ID NO:41).

The final step in the construction of the targeting vector involvedcloning the 8.8 kb SgrAI-AscI fragment containing the “long arm” andRSVLTR-HSVTK gene into the SgrAI and AscI sites of the pSP72-“shortarm”-βgal-PGKneo plasmid. This targeting vector was linearized bydigestion with either AscI or PacI before electroporation into ES cells.

Example 10 Antisense-Mediated Beer Inactivation

17-nucleotide antisense oligonucleotides are prepared in an overlappingformat, in such a way that the 5′ end of the first oligonucleotideoverlaps the translation initiating AUG of the Beer transcript, and the5′ ends of successive oligonucleotides occur in 5 nucleotide incrementsmoving in the 5′ direction (up to 50 nucleotides away), relative to theBeer AUG. Corresponding control oligonucleotides are designed andprepared using equivalent base composition but redistributed in sequenceto inhibit any significant hybridization to the coding mRNA. Reagentdelivery to the test cellular system is conducted through cationic lipiddelivery (P. L. Felgner, Proc. Natl. Acad. Sci. USA 84:7413, 1987). 2 ugof antisense oligonucleotide is added to 100 ul of reduced serum media(Opti-MEM I reduced serum media; Life Technologies, Gaithersburg Md.)and this is mixed with Lipofectin reagent (6 ul) (Life Technologies,Gaithersburg Md.) in the 100 ul of reduced serum media. These are mixed,allowed to complex for 30 minutes at room temperature and the mixture isadded to previously seeded MC3T3E21 or KS483 cells. These cells arecultured and the mRNA recovered. Beer mRNA is monitored using RT-PCR inconjunction with Beer specific primers. In addition, separateexperimental wells are collected and protein levels characterizedthrough western blot methods described in Example 4. The cells areharvested, resuspended in lysis buffer (50 mM Tris pH 7.5, 20 mM NaCl, 1mM EDTA, 1% SDS) and the soluble protein collected. This material isapplied to 10–20% gradient denaturing SDS PAGE. The separated proteinsare transferred to nitrocellulose and the western blot conducted asabove using the antibody reagents described. In parallel, the controloligonucleotides are added to identical cultures and experimentaloperations are repeated. Decrease in Beer mRNA or protein levels areconsidered significant if the treatment with the antisenseoligonucleotide results in a 50% change in either instance compared tothe control scrambled oligonucleotide. This methodology enablesselective gene inactivation and subsequent phenotype characterization ofthe mineralized nodules in the tissue culture model.

SEQUENCES Human BEER cDNA (complete coding region plus 5′ and 3′ UTRs):Sequence ID No. 1AGAGCCTGTGCTACTGGAAGGTGGCGTGCCCTCCTCTGGCTGGTACCATGCAGCTCCCACTGGCCCTGTGTCTCGTCTGCCTGCTGGTACACACAGCCTTCCGTGTAGTGGAGGGCCAGGGGTGGCAGGCGTTCAAGAATGATGCCACGGAAATCATCCCCGAGCTCGGAGAGTACCCCGAGCCTCCACCGGAGCTGGAGAACAACAAGAQCATGAACCGGGCGGAGAACGGAGGGCGGCCTCCCCACCACCCCTTTGAGACCAAAGACGTGTCCGAGTACAGCTGCCGCGAGCTGCACTTCACCCGCTACGTGACCGATGGGCCGTGCCGCAGCGCCAAGCCGGTCACCGAGCTGGTGTGCTCCGGCCAGTGCGGCCCGGCGCGCCTGCTGCCCAACGCCATCGGCCGCGGCAAGTGGTGGCGACCTAGTGGGCCCGACTTCCGCTGCATCCCCGACCGCTACCGCGCGCAGCGCGTGCAGCTGCTGTGTCCCGGTGGTGAGGCGCCGCGCGCGCGCAAGGTGCGCCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACCCGCTTCCACAACCAGTCGGAGCTCAAGGACTTCGGGACCGAGGCCGCTCGGCCGCAGAAGGGCCGGAAGCCGCGGCCCCGCGCCCGGAGCGCCAAAGCCAACCAGGCCGAGCTGGAGAACGCCTACTAGAGCCCGCCCGCGCCCCTCCCCACCGGCGGGCGCCCCGGCCCTGAACCCGCGCCCCACATTTCTGTCCTCTGCGCGTGGTTTGATTGTTTATATTTCATTGTAAATGCCTGCAACCCAGGGCAGGGGGCTGAGACCTTCCAGGCCCTGAGGAATCCCGGGCGCCGGCAAGGCCCCCCTCAGCCCGCCAGCTGAGGGGTCCCACGGGGCAGGGGAGGGAATTGAGAGTCACAGACACTGAGCCACGCAGCCCCGCCTCTGGGGCCGCCTACCTTTGCTGGTCCCACTTCAGAGGAGGCAGAAATGGAAGCATTTTCACCGCCCTGGGGTTTTAAGGGAGCGGTGTGGGAGTGGGAAAGTCCAGGGACTGGTTAAGAAAGTTGGATAAGATTCCCCCTTGCACCTCGCTGCCCATCAGAAAGCCTGAGGCGTGCCCAGAGCACAAGACTGGGGGCAACTGTAGATGTGGTTTCTAGTCCTGGCTCTGCCACTAACTTGCTGTGTAACCTTGAACTACACAATTCTCCTTCGGGACCTCAATTTCCACTTTGTAAAATGAGGGTGGAGGTGGGAATAGGATCTCGAGGAGACTATTGGCATATGATTCCAAGGACTCCAGTGCCTTTTGAATGGGCAGAGGTGAGAGAGAGAGAGAGAAAGAGAGAGAATGAATGCAGTTGCATTGATTCAGTGCCAAGGTCACTTCCAGAATTCAGAGTTGTGATGCTCTCTTCTGACAGCCAAAGATGAAAAACAAACAGAAAAAAAAAAGTAAAGAGTCTATTTATGGCTGACATATTTACGGCTGACAAACTCCTGGAAGAAGCTATGCTGCTTCCCAGCCTGGCTTCCCCGGATGTTTGGCTACCTCCACCCCTCCATCTCAAAGAAATAACATCATCCATTGGGGTAGAAAAGGAGAGGGTCCGAGGGTGGTGGGAGGGATAGAAATCACATCCGCCCCAACTTCCCAAAGAGCAGCATCCCTCCCCCGACCCATAGCCATGTTTTAAAGTCACCTTCCGAAGAGAAGTGAAAGGTTCAAGGACACTGGCCTTGCAGGCCCGAGGGAGCAGCCATCACAAACTCACAGACCAGCACATCCCTTTTGAGACACCGCCTTCTGCCCACCACTCACGGACACATTTCTGCCTAGAAAACAGCTTCTTACTGCTCTTACATGTGATGGCATATCTTACACTAAAAGAATATTATTGGGGGAAAAACTACAAGTGCTGTACATATGCTGAGAAACTGCAGAGCATAATAGCTGCCACCCAAAAATCTTTTTGAAAATCATTTCCAGACAACCTCTTACTTTCTGTGTAGTTTTTAATTGTTAAAAAAAAAAAGTTTTAAACAGAAGCACATGACATATGAAAGCCTGCAGGACTGGTCGTTTTTTTGGCAATTCTTCCACGTGGGACTTGTCCACAAGAATGAAAGTAGTGGTTTTTAAAGAGTTAAGTTACATATTTATTTTCTCACTTAAGTTATTTATGCAAAAGTTTTTCTTGTAGAGAATGACAATGTTAATATTGCTTTATGAATTAACAGTCTGTTCTTCCAGAGTCCAGAGACATTGTTAATAAAGACAATGAATCATGACCGAAAG Human BEERprotein (complete sequence): Sequence ID No. 2MQLPLALCLVCLLVHTAFRVVEGQGWQAFKNDATEIIPELGEYPEPPPELENNKTMNRAENGGRPPHHPFETKDVSEYSCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKCKRLTRFHNQSELRDFGTEAARPQKGRKPRPRARSAKANQAELENAY Human Beer cDNAcontaining Sclerosteosis nonsense mutation: Sequence ID No 3AGAGCCTGTGCTACTGGAAGGTGGCGTGCCCTCCTCTGGCTGGTACCATGCAGCTCCCACTGGCCCTGTGTCTCGTCTGCCTGCTGGTACACACAGCCTTCCGTGTAGTGGAGGGCTAGGGGTGGCAGGCGTTCAAGAATGATGCCACGGAAATCATCCCCGAGCTCGGAGAGTACCCCGAGCCTCCACCGGAGCTGGAGAACAACAAGACCATGAACCCGGCGGAGAACGGAGGGCGGCCTCCCCACCACCCCTTTGAGACCAAAGACGTGTCCGAGTACAGCTGCCGCGAGCTGCACTTCACCCGCTACGTGACCGATGGGCCGTGCCGCAGCGCCAAGCCGGTCACCGAGCTGGTGTGCTCCGGCCAGTGCGGCCCGGCGCGCCTGCTGCCCAACGCCATCGGCCGCGGCAAGTGGTGGCGACCTAGTGGGCCCGACTTCCGCTGCATCCCCGACCGCTACCGCGCGCAGCGCGTGCAGCTGCTGTGTCCCGGTGGTGAGGCGCCGCGCGCGCGCAAGGTGCGCCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACCCGTCTCCACAACCAGTCGGAGCTCAAGGACTTCGGGACCGAGGCCGCTCGGCCGCAGAAGGGCCGGAAGCCGCGGCCCCGCGCCCGGAGCGCCAAAGCCAACCAGGCCGAGCTGGAGAACGCCTACTAGAGCCCGCCCGCGCCCCTCCCCACCGGCGGGCGCCCCGGCCCTGAACCCGCGCCCCACATTTCTGTCCTCTGCGCGTGGTTTGATTGTTTATATTTCATTGTAAATGCCTGCAACCCAGGGCAGGGGGCTGAGACCTTCCAGGCCCTGAGGAATCCCGGGCGCCGGCAAGGCCCCCCTCAGCCCGCCAGCTGAGGGGTCCCACGGGGCAGGGGGGGGAATTGAGAGTCACAGACACTGAGCCACGCAGCCCCGCCTCTGGGGCCGCCTACCTTTGCTGGTCCCACTTCAGAGGAGGCAGAAATGGAAGCATTTTCACCGCCCTGGGGTTTTAAGGGAGCGGTGTGGGAGTGGGAAAGTCCAGGGACTGGTTAAGAAAGTTGGATAAGATTCCCCCTTGCACCTCGCTGCCCATCAGAAAGCCTGAGGCGTGCCCAGAGCACAAGACTGGGGGCAACTGTAGATGTGGTTTCTAGTCCTGGCTCTGCCACTAACTTGCTGTGTAACCTTGAACTACACAATTCTCCTTCGGGACCTCAATTTCCACTTTGTAAAATGAGGGTGGAGGTGGGAATAGGATCTCGAGGAGACTATTGGCATATGATTCCAAGGACTCCAGTGCCTTTTGAATGGGCAGAGGTGAGAGAGAGAGAGAGAAAGAGAGAGAATGAATGCAGTTGCATTGATTCAGTGCCAAGGTCACTTCCAGAATTCAGAGTTGTGATGCTCTCTTCTGACAGCCAAAGATGAAAAACAAACAGAAAAAAAAAAGTAAAGAGTCTATTTATGGCTGACATATTTACGGCTGACAAACTCCTGGAAGAAGCTATGCTGCTTCCCAGCCTGGCTTCCCCGGATGTTTGGCTACCTCCACCCCTCCATCTCAAAGAAATAACATCATCCATTGGCGTAGAAAAGGAGAGGGTCCGAGGGTGGTGGGAGGGATAGAAATCACATCCGCCCCAACTTCCCAAAGAGCAGCATCCCTCCCCCGACCCATAGCCATGTTTTAAAGTCACCTTCCGAAGAGAAGTGAAAGGTTCAAGGACACTGGCCTTGCAGGCCCGAGGGAGCAGCCATCACAAACTCACAGACCAGCACATCCCTTTTGAGACACCGCCTTCTGCCCACCACTCACGGACACATTTCTGCCTAGAAAACAGCTTCTTACTGCTCTTACATGTGATGGCATATCTTACACTAAAAGAATATTATTGGGGGAAAAACTACAAGTGCTGTACATATGCTGAGAAACTGCAGAGCATAATAGCTGCCACCCAAAAATCTTTTTGAAAATCATTTCCAGACAACCTCTTACTTTCTGTGTAGTTTTTAATTGTTAAAAAAAAAAAGTTTTAAACAGAAGCACATGACATATGAAAGCCTGCAGGACTGGTCGTTTTTTTGGCAATTCTTCCACGTGGGACTTGTCCACAAGAATGAAAGTAGTGGTTTTTAAAGAGTTAAGTTACATATTTATTTTCTCACTTAAGTTATTTATGCAAAAGTTTTTCTTGTAGAGAATGACAATGTTAATATTGCTTTATGAATTAACAGTCTGTTCTTCCAGAGTCCAGAGACATTGTTAATAAAGACAATGAATCATGACCGAAAG TruncatedHuman Beer protein from Sclerosteosis: Sequence ID No. 4MQLPLALCLVCLLVHTAFRVVEG* Human BEER cDNA encoding protein variant(V10I): Sequence ID No. 5AGAGCCTGTGCTACTGGAAGGTGGGGTGCCCTCCTCTGGCTGGTACCATGCAGCTCCCACTGGCCCTGTGTCTCATCTGCCTGCTGGTACACACAGCCTTCCGTGTAGTGGAGGGCCAGGGGTGGCAGGCGTTCAAGAATGATGCCACGGAAATCATCCGCGAGCTCGGAGAGTACCCCGAGCCTCCACCGGAGCTGGAGAACAACAAGACCATGAACCGGGCGGAGAACGGAGGGCGGCCTCCCCACCACCCCTTTGAGACCAAAGACGTGTCCGAGTACAGCTGCCGCGAGCTGCACTTCACCCGCTACGTGACCGATGGGCCGTGCCGCAGCGCCAAGCCGGTCACCGAGCTGGTGTGCTCCGGCCAGTGCGGCCCGGCGCGCCTGCTGCCCAACGCCATCGGCCGCGGCAAGTGGTGGCGACCTAGTGGGCCCGACTTCCGCTGCATCCCCGACCGCTACCGCGCGCAGCGCGTGCAGCTGCTGTGTCCCGGTGGTGAGGCGCCGCGCGCGCGCAAGGTGCGCCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACCCGCTTCCACAACCAGTCGGAGCTCAAGGACTTCGGGACCGAGGCCGCTCGGCCGCAGAAGGGCCGGAAGCCGCGCCCCCGCGCCCGGAGCGCCAAAGCCAAACCAGCCGAGCTGGAGAACGCCTACTAGAGCCCGCCCGCGCCCCTCCCCACCGGCGGGCGCCCCGGCCCTGAACCCGCGCCCCACATTTCTGTCCTCTGCGCGTGGTTTGATTGTTTATATTTCATTGTAAATGCCTGCAACCCAGGGCAGGGGGCTGAGACCTTCCAGGCCCTGAGGAATCCCGGGCGCCGGCAAGGCCCCCCTCAGCCCGCCAGCTGAGGGGTCCCACGGGGCAGGGGAGGGAATTGAGAGTCACAGACACTGAGCCACGCAGCCCCGCCTCTGGGGCCGCCTACCTTTGCTGGTCCCACTTCAGAGGAGGCAGAAATGGAAGCATTTTCACCGCCCTGGGGTTTTAAGGGAGCGGTGTGGGAGTGGGAAAGTCCAGGGACTGGTTAAGAAAGTTGGATAAGATTCCCCCTTGCACCTCGCTGCCCATCAGAAAGCCTGAGGCGTGCCCAGAGCACAAGACTGGGGGCAACTGTAGATGTGGTTTCTAGTCCTGGCTCTGCCACTAACTTGCTGTGTAACCTTGAACTACACAATTCTCCTTCGGGACCTCAATTTCCACTTTGTAAAATGAGGGTGGAGGTGGGAATAGGATCTCGAGGAGACTATTGGCATATGATTCCAAGGACTCCAGTGCCTTTTGAATGGGCAGAGGTGAGAGAGAGAGAGAGAAAGAGAGAGAATGAATGCAGTTGCATTGATTCAGTGCCAAGGTCACTTCCAGAATTCAGAGTTGTGATGCTCTCTTCTGACAGCCAAAGATGAAAAACAAACAGAAAAAAAAAAGTAAAGAGTCTATTTATGGCTGACATATTTACGGCTGACAAACTCCTGGAAGAAGCTATGCTGCTTCCCAGCCTGGCTTCCCCGGATGTTTGGCTACCTCCACCCCTCCATCTCAAAGAAATAACATCATCCATTGGGGTAGAAAAGGAGAGGGTCCGAGGGTGGTGGGAGGGATAGAAATCACATCCGCCCCAACTTCCCAAAGAGCAGCATCCCTCCCCCGACCCATAGCCATGTTTTAAAGTCACCTTCCGAAGAGAAGTGAAAGGTTCAAGGACACTGGCCTTGCAGGCCCGAGGGAGCAGCCATCACAAACTCACAGACCAGCACATCCCTTTTGAGACACCGCCTTCTGCCCACCACTCACGGACACATTTCTGCCTAGAAAACAGCTTCTTACTGCTCTTACATGTGATGGCATATCTTACACTAAAAGAATATTATTGGGGGAAAAACTACAAGTGCTGTACATATGCTGAGAAACTGCAGAGCATAATAGCTGCCACCCAAAAATCTTTTTGAAAATCATTTCCAGACAACCTCTTACTTTCTGTGTAGTTTTTAATTGTTAAAAAAAAAAAGTTTTAAACAGAAGCACATGACATATGAAAGCCTGCAGGACTGGTCGTTTTTTTGGCAATTCTTCCACGTGGGACTTGTCCACAAGAATGAAAGTAGTGGTTTTTAAAGAGTTAAGTTACATATTTATTTTCTCACTTAAGTTATTTATGCAAAAGTTTTTCTTGTAGAGAATGACAATGTTAATATTGCTTTATGAATTAACAGTCTGTTCTTCCAGAGTCCAGACACATTGTTAATAAAGACAATGAATCATGACCGAAAG Human BEERprotein variant (V10I): Sequence ID No. 6MQLPLALCLICLLVHTAFRVVEGQGWQAFKNDATEIIRELGEYPEPPPELENNKTMNRAENGGRPPHHPFETKDVSEYSCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKCKRLTRFHNQSELKDFGTEAARPQKGRKPRPRARSAKANQAELENAY Human Beer cDNAencoding protein variant (P38R): Sequence ID No. 7AGAGCCTGTGCTACTGGAAGGTGGCGTGCCCTCCTCTGGCTGGTACCATGCAGCTCCCACTGGCCCTGTGTCTCGTCTGCCTGCTGGTACACACAGCCTTCCGTGTAGTGGAGGGCCAGGGGTGGCAGGCGTCAAGAATGATGCCACCGGAAATCATCCGCGAGCTCGGAGAGTACCCCGAGCCTCCACCGGACCTGGAGAACAACAAGACCATGAACCGGGCGGAGAACGGAGGGCGGCCTCCCCACCACCCCTTTGAGACCAAAGACGTGTCCGAGTACAGCTGCCGCGAGCTGCACTTCACCCGCTACGTGACCGATGGGCCGTGCCGCAGCGCCAAGCCGGTCACCGAGCTGGTGTGCTCCGGCCAGTGCGGCCCGGCGCGCCTGCTGCCCAACGCCATCGGCCGCGGCAAGTGGTGGCGACCTAGTGGGCCCGACTTCCGCTGCATCCCCGACCGCTACCGCGCGCAGCGCGTGCAGCTGCTGTGTCCCGGTGGTGAGGCGCCGCGCGCGCGCAAGGTGCGCCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACCCGCTTCCACAACCAGTCGGAGCTCAAGGACTTCGGGACCGAGGCCGCTCGGCCGCAGAAGGGCCGGAAGCCGCGGCCCCGCGCCCGGAGCGCCAAAGCCAACCAGGCCGAGCTGGAGAACGCCTACTAGAGCCCGCCCGCGCCCCTCCCCACCGGCGGGCGCCCCGGCCCTGAACCCGCGCCCCACATTTCTGTCCTCTGCGCGTGGTTTGATTGTTTATATTTCATTGTAAATGCCTGCAACCCAGGGCAGGGGGCTGAGACCTTCCAGGCCCTGAGGAATCCCGGGCGCCGGCAAGGCCCCCCTCAGCCCGCCAGCTGAGGGGTCCCACGGGGCAGGGGAGGGAATTGAGAGTCACAGACACTGAGCCACGCAGCCCCGCCTCTGGGGCCGCCTACCTTTGCTGGTCCCACTTCAGAGGAGGCAGAAATGGAAGCATTTTCACCGCCCTGGGGTTTTAAGGGAGCGGTGTGGGAGTGGGAAAGTCCAGGGACTGGTTAAGAAAGTTGGATAAGATTCCCCCTTGCACCTCGCTGCCCATCAGAAAGCCTGAGGCGTGCCCAGAGCACAAGACTGGGGGCAACTGTAGATGTGGTTTCTAGTCCTGGCTCTGCCACTAACTTGCTGTGTAACCTTGAACTACACAATTCTCCTTCGGGACCTCAATTTCCACTTTGTAAAATGAGGGTGGAGGTGGGAATAGGATCTCGAGGAGACTATTGGCATATGATTCCAAGGACTCCAGTGCCTTTTGAATGGGCAGAGGTGAGAGAGAGAGAGAGAAAGAGAGAGAATGAATGCAGTTGCATTGATTCAGTGCCAAGGTCACTTCCAGAATTCAGAGTTGTGATGCTCTCTTCTGACAGCCAAAGATGAAAAACAAACAGAAAAAAAAAAGTAAAGAGTCTATTTATGGCTGACATATTTACGGCTGACAAACTCCTGGAAGAAGCTATGCTGCTTCCCAGCCTGGCTTCCCCGGATGTTTGGCTACCTCCACCCCTCCATCTCAAAGAAATAACATCATCCATTGGGGTAGAAAAGGAGAGGGTCCGAGGGTGGTGGGAGGGATAGAAATCACATCCGCCCCAACTTCCCAAAGAGCAGCATCCCTCCCCCGACCCATAGCCATGTTTTAAAGTCACCTTCCGAAGAGAAGTGAAAGGTTCAAGGACACTGGCCTTGCAGGCCCGAGGGAGCAGCCATCACAAACTCACAGACCAGCACATCCCTTTTGAGACACCGCCTTCTGCCCACCACTCACGGACACATTTCTGCCTAGAAAACAGCTTCTTACTGCTCTTACATGTGATGGCATATCTTACACTAAAAGAATATTATTGGGGGAAAAACTACAAGTGCTGTACATATGCTGAGAAACTGCAGAGCATAATAGCTGCCACCCAAAAATCTTTTTGAAAATCATTTCCAGACAACCTCTTACTTTCTGTGTAGTTTTTAATTGTTAAAAAAAAAAAGTTTTAAACAGAAGCACATGACATATGAAAGCCTGCAGGACTGGTCGTTTTTTTGGCAATTCTTCCACGTGGGACTTGTCCACAAGAATGAAAGTAGTGGTTTTTAAAGAGTTAAGTTACATATTTATTTTCTCACTTAAGTTATTTATGCAAAAGTTTTTCTTGTAGAGAATGACAATGTTAATATTGCTTTATGAATTAACAGTCTGTTCTTCCAGAGTCCAGAGACATTGTTAATAAAGACAATGAATCATGACCGAAAG Human Beerprotein variant (P38R): Sequence ID No. 8MQLPLALCLVCLLVHTAFRVVEGQGWQAFKNDATEIIRELGEYPEPPPELENNKTMNRAENGGRPPHHPFETKDVSEYSCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKCKRLTRFHNQSELKDFGTEAARPQKGRKPRPRARSAKANQAELENAY Vervet BEER cDNA(complete coding region): Sequence ID No. 9ATGCAGCTCCCACTGGCCCTGTGTCTTGTCTGCCTGCTGGTACACGCAGCCTTCCGTGTAGTGGAGGGCCAGGGGTGGCAGGCCTTCAAGAATGATGCCACGGAAATCATCCCCGAGCTCGGAGAGTACCCCGAGCCTCCACCGGAGCTGGAGAACAACAAGACCATGAACCGGGCGGAGAATGGAGGGCGGCCTCCCCACCACCCCTTTGAGACCAAAGACGTGTCCGAGTACAGCTGCCGAGAGCTGCACTTCACCCGCTACGTGACCGAtGGGCCGTGCCGCAGCGCCAAGCCAGTCACCGAGTTGGTGTGCTCCGGCCAGTGCGGCCCGGCACGCCTGCTGCCCAACGCCATCGGCCGCGGCAAGTGGTGGCGCCCGAGTGGGCCCGACTTCCGCTGCATCCCCGACCGCTACCGCGCGCACCGTGTGCAGCTGCTGTGTCCCGGTGGTGCCGCGCCGCGCGCGCGCAAGGTGCGCCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACCCGCTTCCACAACCAGTCGGAGCTCAAGGACTTCGGTCCCGAGGCCGCTCGGCCGCAGAAGGGCCGGAAGCCGCGGCCCCGCGCCCGGGGGGCCAAAGCCAATCAGGCCGAGCTGGAGAACGCCTACTAG Vervet BEER protein (complete sequence): Sequence ID No. 10MQLPLALCLVCLLVHAAFRVVEGQGWQAFKNDATEIIPELGEYPEPPPELENNKTMNRAENGGRPPHHPFETKDVSEYSCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGAAPRARKVRLVASCKCKRLTRFHNQSELKDFGPEAARPQKGRKPRPRARGAKANQAELENAY Mouse BEER cDNA(coding region only): Sequence ID No. 11ATGCAGCCCTCACTAGCCCCGTGCCTCATCTGCCTACTTGTGCACGCTGCCTTCTGTGCTGTGGAGGGCCAGGGGTGGCAAGCCTTCAGGAATGATGCCACAGAGGTCATCCCAGGGCTTGGAGAGTACCCCGAGCCTCCTCCTGAGAACAACCAGACCATGAACCGGGCGGAGAATGGAGGCAGACCTCCCCACCATCCCTATGACGCCAAAGGTGTGTCCGAGTACAGCTGCCGCGAGCTGCACTACACCCGCTTCCTGACAGACGGCCCATGCCGCAGCGCCAAGCCGGTCACCGAGTTGGTGTGCTCCGGCCAGTGCGGCCCCGCGCGGCTGCTGCCCAACGCCATCGGGCGCGTGAAGTGGTGGCGCCCGAACGGACCGGATTTCCGCTGCATCCCGGATCGCTACCGCGCGCAGCGGGTGCAGCTGCTGTGCCCCGGGGGCGCGGCGCCGCGCTCGCGCAAGGTGCGTCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACCCGCTTCCACAACCAGTCGGAGCTCAAGGACTTCGGGCCGGAGACCGCGCGGCCGCAGAAGGGTCGCAAGCCGCGGCCCGGCGCCCGGGGAGCCAAAGCCAACCAGGCGGAGCTGGAGAACGCCTACTAGAGMouse BEER protein (complete sequence): Sequence ID No. 12MQPSLAPCLICLLVHAAFCAVEGQGWQAFRNDATEVIPGLGEYPEPPPENNQYMNRAENGGRPPHHPYDAKDVSEYSCRELHYTRFLTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRVKWWRPNGPDFRCIPDRYRAQRVQLLCPGGAAPRSRKVRLVASCKCKRLTRFHNQSELKDFGPETARPQKGRKPRPGARGAKANQAELENAY Rat BEER cDNA(complete coding region plus 5′ UTR): Sequence ID No. 13GAGGACCGAGTGCCCTTCCTCCTTCTGGCACCATGCAGCTCTCACTAGCCCCTTGCCTTGCCTGCCTGCTTGTACATGCAGCCTTCGTTGCTGTGGAGAGCCAGGGGTGGCAAGCCTTCAAGAATGATGCCACAGAAATCATCCCGGGACTCAGAGAGTACCCAGAGCCTCCTCAGGAACTAGAGAACAACCAGACCATGAACCGGGCCGAGAACGGAGGCAGACCCCCCCACCATCCTTATGACACCAAAGACGTGTCCGAGTACAGCTGCCGCGAGCTGCACTACACCCGCTTCGTGACCGACGGCCCGTGCCGCAGTGCCAAGCCGGTCACCGAGTTGGTGTGCTCGGGCCAGTGCGGCCCCGCGCGGCTGCTGCCCAACGCCATCGGGCGCGTGAAGTGGTGGCGCCCGAACGGACCCGACTTCCGCTGCATCCCGGATCGCTACCGCGCGCAGCGGGTGCAGCTGCTGTGCCCCGGCGGCGCGGCGCCGCGCTCGCGCAAGGTGCGTCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACCCGCTTCCACAACCAGTCGGAGCTCAAGGACTTCGGACCTGAGACCGCGCGGCCGCAGAAGGGTCGCAAGCCGCGGCCCCGCGCCCGGGGAGCCAAAGCCAACCAGGCGGAGCTGGALAACGCCTACTAG Rat BEER protein (complete sequence):Sequence ID No. 14MQLSLAPCLACLLVHAAFVAVESQGWQAFKNDATEIIPGLREYPEPPQELENNQTMNRAENGGRPPHHPYDTKDVSEYSCRELHYTRFVTDGPCRSAKPVTELVCSGQCGPARLLPNAEGRVKWWRPNGPDFRCIPDRYRAQRVQLLCPGGAAPRSRKVRLVASCKCKRLTRFHNQSELKDFGPETARPQKGRKPRPRARGAKANQAELENAY Bovine BEER cDNA(partial coding sequence): Sequence ID No. 15AGAATGATGCCACAGAAATCATCCCCGAGCTGGGCGAGTACCCCGAGCCTCTGCCAGAGCTGAACAACAAGACCATGAACCGGGCGGAGAACGGAGGGAGACCTCCCCACCACCCCTTTGAGACCAAAGACGCCTCCGAGTACAGCTGCCGGGAGCTGCACTTCACCCGCTACGTGACCGATGGGCCGTGCCGCAGCGCCAAGCCGGTCACCGAGCTGGTGTGCTCGGGCCAGTGCGGCCCGGCGCGCCTGCTGCCCAACGCCATCGGCCGCGGCAAGTGGTGGCGCCCAAGCGGGCCCGACTTCCGCTGCATCCCCGACCGCTACCGCGCGCAGCGGGTGCAGCTGTTGTGTCCTGGCGGCGCGGCGCCGCGCGCGCGCAAGGTGCGCCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACTCGCTTCCACAACCAGTCCGAGCTCAAGGACTTCGGGCCCGAGGCCGCGCGGCCGCAAACGGGCCGGAAGCTGCGGCCCCGCGCCCGGGGCACCAAAGCCAGCCGGGCCGA Bovine BEER protein(partial sequence -- missing signal sequence and last 6 residues)Sequence ID No. 16NDATEIIPELGEYPEPLPELNNKTMNRAENGGRPPHHPFETKDASEYSCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGAAPRARKVRLVASCKCKRLTRFHNQSELKDFGPEAARPQTGRKLRPRARGTKASRA MluI–AviII restriction fragment used to make mouse Beertransgene Sequence ID No. 17CGCGTTTTGGTGAGCAGCAATATTGCGCTTCGATGAGCCTTGGCGTTGAGATTGATACCTCTGCTGCACAAAAGGCAATCGACCGAGCTGGACCAGCGCATTCGTGACACCGTCTCCTTCGAACTTATTCGCAATGGAGTGTCATTCATCAAGGACNGCCTGATCGCAAATGGTGCTATCCACGCAGCGGCAATCGAAAACCCTCAGCCGGTGACCAATATCTACAACATCAGCCTTGGTATCCTGCGTGATGAGCCAGCGCAGAACAAGGTAACCGTCAGTGCCGATAAGTTCAAAGTTAAACCTGGTGTTGATACCAACATTGAAACGTTGATCGAAAACGCGCTGAAAAACGCTGCTGAATGTGCGGCGCTGGATGTCACAAAGCAAATGGCAGCAGACAAGAAAGCGATGGATGAACTGGCTTCCTATGTCCGCACGGCCATCATGATGGAATGTTTCCCCGGTGGTGTTATCTGGCAGCAGTGCCGTCGATAGTATGCAATTGATAATTATTATCATTTGCGGGTCCTTTCCGGCGATCCGCCTTGTTACGGGGCGGCGACCTCGCGGGTTTTCGCTATTTATGAAAATTTTCCGGTTTAAGGCGTTTCCGTTCTTCTTCGTCATAACTTAATGTTTTTATTTAAAATACCCTCTGAAAAGAAAGGAAACGACAGGTGCTGAAAGCGAGCTTTTTGGCCTCTGTCGTTTCCTTTCTCTGTTTTTGTCCGTGGAATGAACAATGGAAGTCAACAAAAAGCAGAGCTTATCGATGATAAGCGGTCAAACATGAGAATTCGCGGCCGCATAATACGACTCACTATAGGGATCGACGCCTACTCCCCGCGCATGAAGCGGAGGAGCTGGACTCCGCATGCCCAGAGACGCCCCCCAACCCCCAAAGTGCCTGACCTCAGCCTCTACCAGCTCTGGCTTGGGCTTGGGCGGGGTCAAGGCTACCACGTTCTCTTAACAGGTGGCTGGGCTGTCTCTTGGCCGCGCGTCATGTGACAGCTGCCTAGTTCTGCAGTGAGGTCACCGTGGAATGTCTGCCTTCGTTGCCATGGCAACGGGATGACGTTACAATCTGGGTGTGGAGCTTTTCCTGTCCGTGTCAGGAAATCCAAATACCCTAAAATACCCTAGAAGAGGAAGTAGCTGAGCCAAGGCTTTCCTGGCTTCTCCAGATAAAGTTTGACTTAGATGGAAAAAAACAAAATGATAAAGACCCGAGCCATCTGAAAATTCCTCCTAATTGCACCACTAGGAAATGTGTATATTATTGAGCTCGTATGTGTTCTTATTTTAAAAAGAAAACTTTAGTCATGTTATTAATAAGAATTTCTCAGCAGTGGGAGAGAACCAATATTAACACCAAGATAAAAGTTGGCATGATCCACATTGCAGGAAGATCCACGTTGGGTTTTCATGAATGTGAAGACCCCATTTATTAAAGTCCTAAGCTCTGTTTTTGCACACTAGGAAGCGATGGCCGGGATGGCTGAGGGGCTGTAAGGATCTTTCAATGTCTTACATGTGTGTTTCCTGTCCTGCACCTAGGACCTGCTGCCTAGCCTGCAGCAGAGCCAGAGGGGTTTCACATGATTAGTCTCAGACACTTGGGGGCAGGTTGCATGTACTGCATCGCTTATTTCCATACGGAGCACCTACTATGTGTCAAACACCATATGGTGTTCACTCTTCAGAACGGTGGTGGTCATCATGGTGCATTTGCTGACGGTTGGATTGGTGGTAGAGAGCTGAGATATATGGACGCACTCTTCAGCATTCTGTCAACGTGGCTGTGCATTCTTGCTCCTGAGCAAGTGGCTAAACAGACTCACAGGGTCAGCCTCCAGCTCAGTCGCTGCATAGTCTTAGGGAACCTCTCCCAGTCCTCCCTACCTCAACTATCCAAGAAGCCAGGGGGCTTGGCGGTCTCAGGAGCCTGCTTGCTGGGGGACAGGTTGTTGAGTTTTATCTGCAGTAGGTTGCCTAGGCATAGTGTCAGGACTGATGGCTGCCTTGGAGAACACATCCTTTGCCCTCTATGCAAATCTGACCTTGACATGGGGGCGCTGCTCAGCTGGGAGGATCAACTGCATACCTAAAGCCAAGCCTAAAGCTTCTTCGTCCACCTGAAACTCCTGGACCAAGGGGCTTCCGGCACATCCTCTCAGGCCAGTGAGGGAGTCTGTGTGAGCTGCACTTTCCAATCTCAGGGCGTGAGAGGCAGAGGGAGGTGGGGGCAGAGCCTTGCAGCTCTTTCCTCCCATCTGGACAGCGCTCTGGCTCAGCAGCCCATATGAGCACAGGCACATCCCCACCCCACCCCCACCTTTCCTGTCCTGCAGAATTTAGGCTCTGTTCACGGGGGGGGGGGGGGGGGGGCAGTCCTATCCTCTCTTAGGTAGACAGGACTCTGCAGGAGACACTGCTTTGTAAGATACTGCAGTTTAAATTTGGATGTTGTGAGGGGAAAGCGAAGGGCCTCTTTGACCATTCAGTCAAGGTACCTTCTAACTCCCATCGTATTGGGGGGCTACTCTAGTGCTAGACATTGCAGAGAGCCTCAGAACTGTAGTTACCAGTGTGGTAGGATTGATCCTTCAGGGAGCCTGACATGTGACAGTTCCATTCTTCACCCAGTCACCGAACATTTATTCAGTACCTACCCCGTAACAGGCACCGTAGCAGGTACTGAGGGACGGACCACTCAAAGAACTGACAGACCGAAGCCTTGGAATATAAACACCAAAGCATCAGGCTCTGCCAACAGAACACTCTTTAACACTCAGGCCCTTTAACACTCAGGACCCCCACCCCCACCCCAAGCAGTTGGCACTGCTATCCACATTTTACAGAGAGGAAAAACTAGGCACAGGACGATATAAGTGGCTTGCTTAAGCTTGTCTGCATGGTAAATGGCAGGGCTGGATTGAGACCCAGACATTCCAACTCTAGGGTCTATTTTTCTTTTTTCTCGTTGTTCGAATCTGGGTCTTACTGGGTAAACTCAGGCTAGCCTCACACTCATATCCTTCTCCCATGGCTTACGAGTGCTAGGATTCCAGGTGTGTGCTACCATGTCTGACTCCCTGTAGCTTGTCTATACCATCCTCACAACATAGGAATTGTGATAGCAGCACACACACCGGAAGGAGCTGGGGAAATCCCACAGAGGGCTCCGCAGGATGACAGGCGAATGCCTACACAGAAGGTGGGGAAGGGAAGCAGAGGGAACAGCATGGGCGTGGGACCACAAGTCTATTTGGGGAAGCTGCCGGTAACCGTATATGGCTGGGGTGAGGGGAGAGGTCATGAGATGAGGCAGGAAGAGCCACAGCAGGCAGCGGGTACGGGCTCCTTATTGCCAAGAGGCTCGGATCTTCCTCCTCTTCCTCCTTCCGGGGCTGCCTGTTCATTTTCCACCACTGCCTCCCATCCAGGTCTGTGGCTCAGGACATCACCCAGCTGCAGAAACTGGGCATCACCCACGTCCTGAATGGTGCCGAGGGCAGGTCCTTCATGCACGTCAACACCAGTGCTAGCTTCTACGAGGATTCTGGCATCACCTACTTGGGCATCAAGGCCAATGATACGCAGGAGTTCAACCTCAGTGCTTACTTTGAAAGGGCCACAGATTTCATTGACCAGGCGCTGGCCGATAAAAATGGTAAGGAACGTACATTCCGGCACCCATGGAGCGTAAGCCCTCTGGGACCTGCTTCCTCCAAAGAGGCCCCCACTTGAAAAAGGTTCCAGAAAGATCCCAAAATATGCCACCAACTAGGGATTAAGTGTCCTACATGTGAGCCGATGGGGGCCACTGCATATAGTCTGTGCCATAGACATGACAATGGATAATAATATTTCAGACAGAGAGCAGGAGTTAGGTAGCTGTGCTCCTTTCCCTTTAATTGAGTGTGCCCATTTTTTTATTCATGTATGTGTATACATGTGTGTGCACACATGCCATAGGTTGATACTGAACACCGTCTTCAATCGTTCCCCACCCCACCTTATTTTTTGAGGCAGGGTCTCTTCCCTGATCCTGGGGCTCATTGGTTTATCTAGGCTGCTGGCCAGTGAGCTCTGGAGTTCTGCTTTTCTCTACCTCCCTAGCCCTGGGACTGCAGGGGCATGTGCTGGGCCAGGCTTTTATGTCGCGTTGGGGATCTGAACTTAGGTCCCTAGGCCTGAGCACCGTAAAGACTCTGCCACATCCCCAGCCTGTTTGAGCAAGTGAACCATTCCCCAGAATTCCCCCAGTGGGGCTTTCCTACCCTTTTATTGGCTAGGCATTCATGAGTGGTCACCTCGCCAGAGGAATGAGTGGCCACGACTGGCTCAGGGTCAGCAGCCTAGAGATACTGGGTTAAGTCTTCCTGCCGCTCGCTCCCTGCAGCCGCAGACAGAAAGTAGGACTGAATGAGAGCTGGCTAGTGGTCAGACAGGACAGAAGGCTGAGAGGGTCACAGGGCAGATGTCAGCAGAGCAGACAGGTTCTCCCTCTGTGGGGGAGGGGTGGCCCACTGCAGGTGTAATTGGCCTTCTTTGTGCTCCATAGAGGCTTCCTGGGTACACAGCAGCTTCCCTGTCCTGGTGATTCCCAAAGAGAACTCCCTACCACTGGACTTACAGAAGTTCTATTGACTGGTGTAAGGGTTCAACAGCTTTGGCTCTTGGTGGACGGTGCATACTGCTGTATCAGCTCAAGAGCTCATTCACGAATGAACACACACACACACACACACACACACACACACACAAGCTAATTTTGATATGCCTTAACTAGCTCAGTGACTGGGCATTTCTGAACATCCCTGAAGTTAGCACACATTTCCCTCTGGTGTTCCTGGCTTAACACCTTCTAAATCTATATTTTATCTTTGCTGCCCTGTTACCTTCTGAGAAGCCCCTAGGGCCACTTCCCTTCGCACCTACATTGCTGGATGGTTTCTCTCCTGCAGCTCTTAAATCTGATCCCTCTGCCTCTGAGCCATGGGAACAGCCCAATAACTGAGTTAGACATAAAAACGTCTCTAGCCAAAACTTCAGCTAAATTTAGACAATAAATCTTACTGGTTGTGGAATCCTTAAGATTCTTCATGACCTCCTTCACATGGCACGAGTATGAAGCTTTATTACAATTGTTTATTGATCAAACTAACTCATAAAAAGCCAGTTGTCTTTCACCTGCTCAAGGAAGGAACAAAATTCATCCTTAACTGATCTGTGCACCTTGCACAATCCATACGAATATCTTAAGAGTACTAAGATTTTGGTTGTGAGAGTCACATGTTACAGAATGTACAGCTTTGACAAGGTGCATCCTTGGGATGCCGAAGTGACCTGCTGTTCCAGCCCCCTACCTTCTGAGGCTGTTTTGGAACCAATGCTCTGGAAGCAACTTTAGGAGGTAGGATGCTGGAACAGCGGGTCACTTCAGCATCCCGATGACGAATCCCGTCAAAGCTGTACATTCTGTAACAGACTGGGAAAGCTGCAGACTTTAAGGCCAGGGCCCTATGGTCCCTCTTAATCCCTGTCACAGGGAACCCGAGCCCTTCTCCTCCAGCCGTTCTGTGCTTCTCACTCTGGATAGATGGAGAACACGGCCTTGCTAGTTAAAGGAGTGAGGCTTCACCCTTCTCACATGGCAGTGGTTGGTCATCCTCATTCAGGGAACTCTGGGGCATTCTGCCTTTACTTCCTCTTTTTGGACTACAGGGAATATATGCTGACTTGTTTTGACCTTGTGTATGGGGAGACTGGATCTTTGGTCTGGAATGTTTCCTGCTAGTTTTTCCCCATCCTTTGGCAAACCCTATCTATATCTTACCACTAGGCATAGTGGCCCTCGTTCTGGAGCCTGCCTTCAGGCTGGTTCTCGGGGACCATGTCCCTGGTTTCTCCCCAGCATATGGTGTTCACAGTGTTCACTGCGGGTGGTTGCTGAACAAAGCGGGGATTGCATCCCAGAGCTCCGGTGCCTTGTGGGTACACTGCTAAGATAAAATGGATACTGGCCTCTCTCTGACCACTTGCAGAGCTCTGGTGCCTTGTGGGTACACTGCTAAGATAAAATGGATACTGGCCTCTCTCTATCCACTTGCAGGACTCTAGGGAACAGGAATCCATTACTGAGAAAACCAGGGGCTAGGAGCAGGGAGGTAGCTGGGCAGCTGAAGTGCTTGGCGACTAACCAATGAATACCAGAGTTTGGATCTCTAGAATACTCTTAAAATCTGGGTGGGCAGAGTGGCCTGCCTGTAATCCCAGAACTCGGGAGGCGGAGACAGGGAATCATCAGAGCAAACTGGCTAACCAGAATAGCAAAACACTGAGCTCTGGGCTCTGTGAGAGATCCTGCCTTAACATATAAGAGAGAGAATAAAACATTGAAGAAGACAGTAGATGCCAATTTTAAGCCCGCACATGCACATGGACAAGTGTGCGTTTGAACACACATATGCACTCATGTGAACCAGGCATGCACACTCGGGCTTATCACACACATAATTTGAAAGAGAGAGTGAGAGAGGAGAGTGCACATTAGAGTTCACAGGAAAGTGTGAGTGAGCACACCCATGCACACAGACATGTGTGCCAGGGAGTAGGAAAGGAGCCTGGGTTTGTGTATAAGAGGGAGCCATCATGTGTTTCTAAGGAGGGCGTGTGAAGGAGGCGTTGTGTGGGCTGGGACTGGAGCATGGTTGTAACTGAGCATGCTCCCTGTGGGAAACAGGAGGGTGGCCACCCTGCAGAGGGTCCCACTGTCCAGCGGGATCAGTAAAAGCCCCTGCTGAGAACTTTAGGTAATAGCCAGAGAGAGAAAGGTAGGAAAGTGGGGGGACTCCCATCTCTGATGTAGGAGGATCTGGGCAAGTAGAGGTGCGTTTGAGGTAGAAAGAGGGGTGCAGAGGAGATGCTCTTAATTCTGGGTCAGCAGTTTCTTTCCAAATAATGCCTGTGAGGAGGTGTAGGTGGTGGCCATTCACTCACTCAGCAGAGGGATGATGATGCCCGGTGGATGCTGGAAATGGCCGAGCATCAAGGGTGGCTCTGGAAGAACTCCATCTTTCAGAAGGAGAGTGGATCTGTGTATGGCCAGCGGGGTCACAGGTGCTTGGGGCCCCTGGGGGACTCCTAGCACTGGGTGATGTTTATCGAGTGCTCTTGTGTGCCAGGCACTGGCCTGGGGCTTTGTTTCTGTCTCTGTTTTGTTTCGTTTTTTGAGACAGACTCTTGCTATGTATCCGTGTCAATCTTGGAATCTCACTGCATAGCCCAGGCTGCGGAGAGAGGGGAGGGCAATAGGCCTTGTAAGCAAGCCACACTTCAGAGACTAGACTCCACCCTGCGAATGATGACAGGTCAGAGCTGAGTTCCGGAAGATTTTTTTTCCAGCTGCCAGGTGGAGTGTGGAGTGGCAGCTAGCGGCAAGGGTAGAGGGCGAGCTCCCTGTGCAGGAGAAATGCAAGCAAGAGATGGCAAGCCAGTGAGTTAAGCATTCTGTGTGGGGAGCAGGTGGATGAAGAGAGAGGCTGGGCTTTCGCCTCTGGGGGGGGGGTGAGGGGTGGGGATGAGGTGAGAGGAGGGCAGCTCCCTGCAGTGTGATGAGATTTTTCCTGACAGTGACCTTTGGCCTCTCCCTCCCCCACTTCCCTTCTTTCCTTTCTTCCCACCATTGCTTTCCTTGTCCTTGAGAAATTCTGAGTTTCCACTTCACTGGTGATGCAGACGGAAACAGAAGCCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTGTGTGTATGTGTGTGTGTGTGTTTGTGTGTATGTGTGTCAGTGGGAATGGCTCATAGTCTGCAGGAAGGTGGGCAGGAAGGAATAAGCTGTAGGCTGAGGCAGTGTGGGATGCAGGGAGAGAGGAGAGGAGGGATACCAGAGAAGGAAATTAAGGGAGCTACAAGAGGGCATTGTTGGGGTGTGTGTGTGTGTGTGTTGTTTATATTTGTATTGGAAATACATTCTTTTAAAAAATACTTATCCATTTATTTATTTTTATGTGCACGTGTGTGTGCCTGCATGAGTTCATGTGTGCCACGTGTGTGCGGGAACCCTTGGAGGCCACAAGGGGGCATCTGATCCCCTGGAACTGGAGTTGGAGGAGGTTGTGAGTCCCCTGACATGTTTGCTGGGAACTGAACCCCGGTCCTATGCAAGAGCAGGAAGTGCAGTTATCTGCTGAGCCATCTGTCCAGTCCTGAAATCCATTCTCTTAAAATACACGTGGCAGAGACATGATGGGATTTACGTATGGATTTAATGTGGCGGTCATTAAGTTCCGGCACAGGCAAGCACCTGTAAAGCCATCACCACAACCGCAACAGTGAATGTGACCATCACCCCCATGTTCTTCATGTCCCCTGTCCCCTCCATCCTCCATTCTCAAGCACCTCTTGCTCTGCCTCTGTCGCTGGAGAACAGTGTGCATCTGCACACTCTTATGTCAGTGAAGTCACACAGCCTGCACCCCTTCCTGGTCTGAGTATTTGGGTTCTGACTCTGCTATCACACACTACTGTACTGCATTCTCTCGCTCTCTTTTTTTAAACATATTTTTATTTGTTTGTGTGTATGCACATGTGCCACATGTGTACAGATACTATGGAGGCCAGAAGAGGCCATGGCCGTCCCTGGAGCTGGAGTTACAGGCAGCGTGTGAGCTGCCTGGTGTGGGTGCTGGGAACCAAACTTGAATCTAAAGCAAGCACTTTTAACTGCTGAGGCAGCTCTCAGTACCCTTCTTCATTTCTCCGCCTGGGTTCCATTGTATGGACACATGTAGCTAGAATATCTTGCTTATCTAATTATGTACATTGTTTTGTGCTAAGAGAGAGTAATGCTCTATACCCTGAGCTGGCCTCAACCTTGCCATCCTCCTGCCTCAGCCTCCTCCTCCTGAGTGCTAGGATGACAGGCGAGTGGTAACTTACATGGTTTCATGTTTTGTTCAAGACTGAAGGATAACATTCATACAGAGAAGGTCTGGGTCACAAAGTGTGCAGTTCACTGAATGGCACAACCCGTGATCAAGAAACAAAACTCAGGGGCTGGAGAGATGGCACTGACTGCTCTTCCAGAGGTCCGGAGTTCAATTCCCAGCAACCACATGGTGGCTCACAGCCATCTATAACGAGATCTGACGCCCTCTTCTGGTGTGTCTGAAGACAGCTACAGTGTACTCACATAAAATAAATAAATCTTTAAAACACACACACACACACAATTACCACCCCAGAAAGCCCACTCCATGTTCCCTCCCACGTCTCTGCCTACAGTACTCCCAGGTTACCACTGTTCAGGCTTCTAACAACCTGGTTTACTTGGGCCTCTTTTCTGCTCTGTGGAGCCACACATTTGTGTGCCTCATACACGTTCTTTCTAGTAAGTTGCATATTACTCTGAGTTTTTACATGTATTTATTTATTGTAGTTGTGTGTGCGTGTGGGCCCATGCATGGCACAGTGTGTGGGGATGTCAGAGTATTGTGAACAGGGGACAGTTCTTTTCTTCAATCATGTGGGTTCCAGAGGTTGAACTCAGGTCATCATGTGTGGCAGCAAATGCCTTTACCCACTGAGACATCTCCATATTCTTTTTTTTTCCCCTGAGGTGGGGGCTTGTTCCATAGCCCAAACTGGCTTTGCACTTGCAGTTCAAAGTGACTCCCTGTCTCCACCTCTTAGAGTATTGGAATTACGATGTGTACTACCACACCTGACTGGATCATTAATTCTTTCATGGGGGCGGGGAAGCGCACATGCTGCAGGTGAAGGGATGACTGGACTGGACATGAGCGTGGAAGCCAGAGAACAGCTTCAGTCTAATGCTCTCCCAACTGAGCTATTTCGGTTTGCCAGAGAACAACTTACAGAAAGTTCTCAGTGCCATGTGGATTCGGGGTTGGAGTTCAACTCATCAGCTTGACATTGGCTCCTCTACCCACTGAGCCTTCTCACTACTCTCTACGTAGATCATTAATTCTTTTTTAAAAAGACTTATTAGGGGGCTGGAGAGATGGCTCAGCCGTTAAGAGCACCGAATGCCCTTCCAGAGGTCCTGAGTTCAATTCCCAGCATGCCATTGCTGGCCAGTAGGGGGCGCAGGTGTTCAACGTGAGTAGCTGTTGCCAGTTTTCCGCGGTGGAGAACCTCTTGACACCCTGCTGTCCCTGGTCATTCTGGGTGGGTGCATGGTGATATGCTTGTTGTATGGAAGACTTTGACTGTTACAGTGAAGTTGGGCTTCCACAGTTACCACGTCTCCCCTGTTTCTTGCAGGCCGGGTGCTTGTCCATTGCCGCGAGGGCTACAGCCGCTCCCCAACGCTAGTTATCGCCTACCTCATGATGCGGCAGAAGATGGACGTCAAGTCTGCTCTGAGTACTGTGAGGCAGAATCGTGAGATCGGCCCCAACGATGGCTTCCTGGCCCAACTCTGCCAGCTCAATGACAGACTAGCCAAGGAGGGCAAGGTGAAACTCTAGGGTGCCCACAGCCTCTTTTGCAGAGGTCTGACTGGGAGGGCCCTGGCAGCCATGTTTAGGAAACACAGTATACCCACTCCCTGCACCACCAGACAGGTGCCCACATCTGTCCCACTCTGGTCCTCGGGGGCCACTCCACCCTTAGGGAGCACATGAAGAAGCTCCCTAAGAAGTTCTGCTCCTTAGCCATCCTTTCCTGTAATTTATGTCTCTCCCTGAGGTGAGGTTCAGGTTTATGTCCCTGTCTGTGGCATAGATACATCTCAGTGACCCAGGGTGGGAGGGCTATCAGGGTGCATGGCCCGGGACACGGGCACTCTTCATGACCCCTCCCCCACCTGGGTTCTTCCTGTGTGGTCCAGAACCACGAGCCTGGTAAAGGAACTATGCAAACACAGGCCCTGACCTCCCCATGTCTGTTCCTGGTCCTCACAGCCCGACACGCCCTGCTGAGGCAGACGAATGACATTAAGTTCTGAAGCAGAGTGGAGATAGATTAGTGACTAGATTTCCAAAAAGAAGGAAAAAAAAGGCTGCATTTTAAAATTATTTCCTTAGAATTAAAGATACTACATAGGGGCCCTTGGGTAAGCAAATCCATTTTTCCCAGAGGCTATCTTGATTCTTTGGAATGTTTAAAGTGTGCCTTGCCAGAGAGCTTACGATCTATATCTGCTGCTTCAGAGCCTTCCCTGAGGATGGCTCTGTTCCTTTGCTTGTTAGAAGAGCGATGCCTTGGGCAGGGTTTCCCCCTTTTCAGAATACAGGGTGTAAAGTCCAGCCTATTACAAACAAACAAACAAACAAACAAACAAAGGACCTCCATTTGGAGAATTGCAAGGATTTTATCCTGAATTATAGTGTTGGTGAGTTCAAGTCATCACGCCAAGTGCTTGCCATCCTGGTTGCTATTCTAAGAATAATTAGGAGGAGGAACCTAGCCAATTGCAGCTCATGTCCGTGGGTGTGTGCACGGGTGCATATGTTGGAAGGGGTGCCTGTCCCCTTGGGGACAGAAGGAAAATGAAAGGCCCCTCTGCTCACCCTGGCCATTTACGGGAGGCTCTGCTGGTTCCACGGTGTCTGTGCAGGATCCTGAAACTGACTCGCTGGACAGAAACGAGACTTGGCGGCACCATGAGAATGGAGAGAGAGAGAGCAAAGAAAGAAACAGCCTTTAAAAGAACTTTCTAAGGGTGGTTTTTGAACCTCGCTGGACCTTGTATGTGTGCACATTTGCCAGAGATTGAACATAATCCTCTTGGGACTTCACGTTCTCATTATTTGTATGTCTCCGGGGTCACGCAGAGCCGTCAGCCACCACCCCAGCACCCGGCACATAGGCGTCTCATAAAAGCCCATTTTATGAGAACCAGAGCTGTTTGAGTACCCCGTGTATAGAGAGAGTTGTTGTCGTGGGGCACCCGGATCCCAGCAGCCTGGTTGCCTGCCTGTAGGATGTCTTACAGGAGTTTGCAGAGAAACCTTCCTTGGAGGGAAAGAAATATCAGGGATTTTTGTTGAATATTTCAAATTCAGCTTTAAGTGTAAGACTCAGCAGTGTTCATGGTTAAGGTAAGGAACATGCCTTTTCCAGAGCTGCTGCAAGAGGCAGGAGAAGCAGACCTGTCTTAGGATGTCACTCCCAGGGTAAAGACCTCTGATCACAGCAGGAGCAGAGCTGTGCAGCCTGGATGGTCATTGTCCCCTATTCTGTGTGACCACAGCAACCCTGGTCACATAGGGCTGGTCATCCTTTTTTTTTTTTTTTTTTTTTTTTTTTGGCCCAGAATGAAGTGACCATAGCCAAGTTGTGTACCTCAGTCTTTAGTTTCCAAGCGGCTCTCTTGCTCAATACAATGTGCATTTCAAAATAACACTGTAGAGTTGACAGAACTGGTTCATGTGTTATGAGAGAGGAAAAGAGAGGAAAGAACAAAACAAAACAAAACACCACAAACCAAAAACATCTGGGCTAGCCAGGCATGATTGCAATGTCTACAGGCCCAGTTCATGAGAGGCAGAGACAGGAAGACCGCCGAAAGGTCAAGGATAGCATGGTCTACGTATCGAGACTCCAGCCAGGGCTACGGTCCCAAGATCCTAGGTTTTGGATTTTGGGCTTTGGTTTTTGAGACAGGGTTTCTCTGTGTAGCCCTGGCTGTCCTGGAACTCGCTGTGTAGACCAGGCTGGCCTCAAACTTAGAGATCTGCCTGACTCTGCCTTTGAGGGCTGGGACGAATGCCACCACTGCCCAACTAAGATTCCATTAAAAAAAAAAAAAGTTCAAGATAATTAAGAGTTGCCAGCTCGTTAAAGCTAAGTAGAAGCAGTCTCAGGCCTGCTGCTTGAGGCTGTTCTTGGCTTGGACCTGAAATCTGCCCCCAACAGTGTCCAAGTGCACATGACTTTGAGCCATCTCCAGAGAAGGAAGTGAAAATTGTGGCTCCCCAGTCGATTGGGACACAGTCTCTCTTTGTCTAGGTAACACATGGTGACACATAGCATTGAACTCTCCACTCTGAGGGTGGGTTTCCCTCCCCCTGCCTCTTCTGGGTTGGTCACCCCATAGGACAGCCACAGGACAGTCACTAGCACCTACTGGAAACCTCTTTGTGGGAACATGAAGAAAGAGCCTTTGGGAGATTCCTGGCTTTCCATTAGGGCTGAAAGTACAACGGTTCTTGGTTGGCTTTGCCTCGTGTTTATAAAACTAGCTACTATTCTTCAGGTAAAATACCGATGTTGTGGAAAAGCCAACCCCGTGGCTGCCCGTGAGTAGGGGGTGGGGTTGGGAATCCTGGATAGTGTTCTATCCATGGAAAGTGGTGGAATAGGAATTAAGGGTGTTCCCCCCCCCCCCAACCTCTTCCTCAGACCCAGCCACTTTCTATGACTTATAAACATCCAGGTAAAAATTACAAACATAAAAATGGTTTCTCTTCTCAATCTTCTAAAGTCTGCCTGCCTTTTCCAGGGGTAGGTCTGTTTCTTTGCTGTTCTATTGTCTTGAGAGCACAGACTAACACTTACCAAATGAGGGAACTCTTGGCCCATACTAAGGCTCTTCTGGGCTCCAGCACTCTTAAGTTATTTTAAGAATTCTCACTTGGCCTTTAGCACACCCGCCACCCCCAAGTGGGTGTGGATAATGCCATGGCCAGCAGGGGGCACTGTTGAGGCGGGTGCCTTTCCACCTTAAGTTGCTTATAGTATTTAAGATGCTAAATGTTTTAATCAAGAGAAGCACTGATCTTATAATACGAGGATAAGAGATTTTCTCACAGGAAATTGTCTTTTTCATAATTCTTTTACAGGCTTTGTCCTGATCGTAGCATAGAGAGAATAGCTGGATATTTAACTTGTATTCCATTTTCCTCTGCCAGCGTTAGGTTAACTCCGTAAAAAGTGATTCAGTGGACCGAAGAGGCTCAGAGGGCAGGGGATGGTGGGGTGAGGCAGAGCACTGTCACCTGCCAGGCATGGGAGGTCCTGCCATCCGGGAGGAAAAGGAAAGTTTAGCCTCTAGTCTACCACCAGTGTTAACGCACTCTAAAGTTGTAACCAAAATAAATGTCTTACATTACAAAGACGTCTGTTTTGTGTTTCCTTTTGTGTGTTTGGGCTTTTTATGTGTGCTTTATAACTGCTGTGGTGGTGCTGTTGTTAGTTTTGAGGTAGGATCTCAGGCTGGCCTTGAACTTCTGATCGCCTGCCCCTGCCCCTGCCCCTGCCCCTGTCCCTGCCTCCAAGTGCTAGGACTAAAAGCACATGCCACCACACCAGTACAGCATTTTTCTAACATTTAAAAATAATCACCTAGGGGCTGGAGAGAGGGTTCCAGCTAAGAGTGCACACTGCTCTTGGGTAGGACCTGAGTTTAGTTCCCAGAACCTATACTGGGTGGCTCCAGGTCCAGAGGATCCAGGACCTCTGGCCTCCATGGGCATCTGCTCTTAGCACATACCCACATACAGATACACACATAAAAATAAAATGAAGCCTTTAAAAACCTCCTAAAACCTAGCCCTTGGAGGTACGACTCTGGAAAGCTGGCATACTGTGTAAGTCCATCTCATGGTGTTCTGGCTAACGTAAGACTTACAGAGACAGAAAAGAACTCAGGGTGTGCTGGGGGTTGGGATGGAGGAAGAGGGATGAGTAGGGGGAGCACGGGGAACTTGGGCAGTGAAAATTCTTTGCAGGACACTAGAGGAGGATAAATACCAGTCATTGCACCCACTACTGGACAACTCCAGGGAATTATGCTGGGTGAAAAGAGAAGGCCCCAGGTATTGGCTGCATTGGCTGCATTTGCGTAACATTTTTTTAAATTGAAAAGAAAAAGATGTAAATCAAGGTTAGATGAGTGGTTGCTGTGAGCTGAGAGCTGGGGTGAGTGAGACATGTGGACAACTCCATCAAAAAGCGACAGAAAGAACGGGCTGTGGTGACAGCTACCTCTAATCTCCACCTCCGGGAGGTGATCAAGGTTAGCCCTCAGCTAGCCTGTGGTGCATGAGACCCTGTTTCAAAAACTTTAATAAAGAAATAATGAAAAAAGACATCAGGGCAGATCCTTGGGGCCAAAGGCGGACAGGCGAGTCTCGTGGTAAGGTCGTGTAGAAGCGGATGCATGAGCACGTGCCGCAGGCATCATGAGAGAGCCCTAGGTAAGTAAGGATGGATGTGAGTGTGTCGGCGTCGGCGCACTGCACGTCCTGGCTGTGGTGCTGGACTGGCATCTTTGGTGAGCTGTGGAGGGGAAATGGGTAGGGAGATCATAAAATCCCTCCGAATTATTTCAAGAACTGTCTATTACAATTATCTCAAAATATTAAAAAAAAAGAAGAATTAAAAAACAAAAAACCTATCCAGGTGTGGTGGTGTGCACCTATAGCCACGGGCACTTGGAAAGCTGGAGCAAGAGGATGGCGAGTTTGAAGGTATCTGGGGCTGTACAGCAAGACCGTCGTCCCCAAACCAAACCAAACAGCAAACCCATTATGTCACACAAGAGTGTTTATAGTGAGCGGCCTCGCTGAGAGCATGGGGTGGGGGTGGGGGTGGGGGACAGAAATATCTAAACTGCAGTCAATAGGGATCCACTGAGACCCTGGGGCTTGACTGCAGCTTAACCTTGGGAAATGATAAGGGTTTTGTGTTGAGTAAAAGCATCGATTACTGACTTAACCTCAAATGAAGAAAAAAAAAAAAAAAAAACAACAAAAGCCAAACCAAGGGGCTGGTGAGATGGCTCAGTGGGTAAGAGCACCCGACTGCTCTTCCGAAGGTCCAGAGTTCAAATCCCAGCAACCACATCGTGGCTCACAACCATCTGTAACGAGATATGATGCCCTCTTCTGGTGTGTCTGAAGACAGCTACAGTGTACTTACATATAATAAATAAATCTTAAAAAAAAAAAAAAAAAAAAAAGCCAAACCGAGCAAACCAGGCCCCCAAACAGAAGGCAGGCACGACGGCAGGCACCACGAGCCATCCTGTGAAAAGGCAGGGCTACCCATGGGCCGAGGAGGGTCCAGAGAGATAGGCTGGTAAGCTCAGTTTCTCTGTATACCCTTTTTCTTGTTGACACTACTTCAATTACAGATAAAATAACAAATAAACAAAATCTAGAGCCTGGCCACTCTCTGCTCGCTTGATTTTTCCTGTTACGTCCAGCAGGTGGCGGAAGTGTTCCAAGGACAGATCGCATCATTAAGGTGGCCAGCATAATCTCCCATCAGCAGGTGGTGCTGTGAGAACCATTATGGTGCTCACAGAATCCCGGGCCCAGGAGCTGCCCTCTCCCAAGTCTGGAGCAATAGGAAAGCTTTCTGGCCCAGACAGGGTTAACAGTCCACATTCCAGAGCAGGGGAAAAGGAGACTGGAGGTCACAGACAAAAGGGCCAGCTTCTAACAACTTCACAGCTCTGGTAGGAGAGATAGATCACCCCCAACAATGGCCACAGCTGGTTTTGTCTGCCCCGAAGGAAACTGACTTAGGAAGCAGGTATCAGAGTCCCCTTCCTGAGGGGACTTCTGTCTGCCTTGTAAAGCTGTCAGAGCAGCTGCATTGATGTGTGGGTGACAGAAGATGAAAAGGAGGACCCAGGCAGATCGCCACAGATGGACCGGCCACTTACAAGTCGAGGCAGGTGGCAGAGCCTTGCAGAAGCTCTGCAGGTGGACGACACTGATTCATTACCCAGTTAGCATACCACAGCGGGCTAGGCGGACCACAGCCTCCTTCCCAGTCTTCCTCCAGGGCTGGGGAGTCCTCCAACCTTCTGTCTCAGTGCAGCTTCCGCCAGCCCCTCCTCCTTTTGCACCTCAGGTGTGAACCCTCCCTCCTCTCCTTCTCCCTGTGGCATGGCCCTCCTGCTACTGCAGGCTGAGCATTGGATTTCTTTGTGCTTAGATAGACCTGAGATGGCTTACTGATTTATATATATATATCCATCCCTTGGATCTTACATCTAGGACCCAGAGCTGTTTGTGATACCATAAGAGGCTGGGGAGATGATATGGTAAGAGTGCTTGCTGTACAAGCATGAAGACATGAGTTCGAATCCCCAGCAACCATGTGGAAAAATAACCTTCTAACCTCAGAGTTGAGGGGAAAGGCAGGTGGATTCTGGGGGCTTACTGGCCAGCTAGCCAGCCTAACCTAAATGTCTCAGTCAGAGATCCTGTCTCAGGGAATAACTTGGGAGAATGACTGAGAAAGACACCTCCTCAGGTCTCCCATGCACCCACACAGACACACGGGGGGGGGGTAATGTAATAAGCTAAGAAATAATGAGGGAAATGATTTTTTGCTAAGAAATGAAATTCTGTGTTGGCCGCAAGAAGCCTGGCCAGGGAAGGAACTGCCTTTGGCACACCAGCCTATAAGTCACCATGAGTTCCCTGGCTAAGAATCACATGTAATGGAGCCCAGGTCCCTCTTGCCTGGTGGTTGCCTCTCCCACTGGTTTTGAAGAGAAATTCAAGAGAGATCTCCTTGGTCAGAATTGTAGGTGCTGAGCAATGTGGAGCTGGGGTCAATGGGATTCCTTTAAAGGCATCCTTCCCAGGGCTGGGTCATACTTCAATAGTAGGGTGCTTGCACAGCAAGCGTGAGACCCTAGGTTAGAGTCCCCAGAATCTGCCCCCAACCCCCCAAAAAGGCATCCTTCTGCCTCTGGGTGGGTGGGGGGAGCAAACACCTTTAACTAAGACCATTAGCTGGCAGGGGTAACAAATGACCTTGGCTAGAGGAATTTGGTCAAGCTGGATTCCGCCTTCTGTAGAAGCCCCACTTGTTTCCTTTGTTAAGCTGGCCCACAGTTTGTTTTGAGAATGCCTGAGGGGCCCAGGGAGCCAGACAATTAAAAGCCAAGCTCATTTTGATATCTGAAAACCACAGCCTGACTGCCCTGCCCGTGGGAGGTACTGGGAGAGCTGGCTGTGTCCCTGCCTCACCAACGCCCCCCCCCCCAACACACACTCCTCGGGTCACCTGGGAGGTGCCAGCAGCAATTTGGAAGTTTACTGAGCTTGAGAAGTCTTGGGAGGGCTGACGCTAAGCACACCCCTTCTCCACCCCCCCCCACCCCACCCCCGTGAGGAGGAGGGTGAGGAAACATGGGACCAGCCCTGCTCCAGCCCGTCCTTATTGGCTGGCATGAGGCAGAGGGGGCTTTAAAAAGGCAACCGTATCTAGGCTGGACACTGGAGCCTGTGCTACCGAGTGCCCTCCTCCACCTGGCAGCATGCAGCCCTCACTAGCCCCGTGCCTCATCTGCCTACTTGTGCACGCTGCCTTCTGTGCTGTGGAGGGCCAGGGGTGGCAAGCCTTCAGGAATGATGCCACAGAGGTCATCCCAGGGCTTGGAGAGTACCCCGAGCCTCCTCCTGAGAACAACCAGACCATGAACCGGGCGGAGAATGGAGGCAGACCTCCCCACCATCCCTATGACGCCAAAGGTACGGGATGAAGAAGCACATTAGTGGGGGGGGGGGTCCTGGGAGGTGACTGGGGTGGTTTTAGCATCTTCTTCAGAGGTTTGTGTGGGTGGCTAGCCTCTGCTACATCAGGGCAGGGACACATTTGCCTGGAAGAATACTAGCACAGCATTAGAACCTGGAGGGCAGCATTGGGGGGCTGGTAGAGAGCACCCAAGGCAGGGTGGAGGCTGAGGTCAGCCGAAGCTGGCATTAACACGGGCATGGGCTTGTATGATGGTCCAGAGAATCTCCTCCTAAGGATGAGGACACAGGTCAGATCTAGCTGCTGACCAGTGGGGAAGTGATATGGTGAGGCTGGATGCCAGATGCCATCCATGGCTGTACTATATCCCACATGACCACCACATGAGGTAAAGAAGGCCCCAGCTTGAAGATGGAGAAACCGAGAGGCTCCTGAGATAAAGTCACCTGGGAGTAAGAAGAGCTGAGACTGGAAGCTGGTTTGATCCAGATGCAAGGCAACCCTAGATTGGGTTTGGGTGGGAACCTGAAGCCAGGAGGAATCCCTTTAGTTCCCCCTTGCCCAGGGTCTGCTCAATGAGCCCAGAGGGTTAGCATTAAAAGAACAGGGTTTGTAGGTGGCATGTGACATGAGGGGCAGCTGAGTGAAATGTCCCCTGTATGAGCACAGGTGGCACCACTTGCCCTGAGCTTGCACCCTGACCCCAGCTTTGCCTCATTCCTGAGGACAGCAGAAACTGTGGAGGCAGAGCCAGCACAGAGAGATGCCTGGGGTGGGGGTGGGGGTATCACGCACGGAACTAGCAGCAATGAATGGGGTGGGGTGGCAGCTGGAGGGACACTCCAGAGAAATGACCTTGCTGGTCACCATTTGTGTGGGAGGAGAGCTCATTTTCCAGCTTGCCACCACATGCTGTCCCTCCTGTCTCCTAGCCAGTAAGGGATGTGGAGGAAAGGGCCACCCCAAAGGAGCATGCAATGCAGTCACGTTTTTGCAGAGGAAGTGCTTGACCTAAGGGCACTATTCTTGGAAAGCCCCAAAACTAGTCCTTCCCTGGGCAAACAGGCCTCCCCCACATACCACCTCTGCAGGGGTGAGTAAATTAAGCCAGCCACAGAAGGGTGGCAAGGCCTACACCTCCCCCCTGTTGTGCCCCCCCCCCCCCCGTGAAGGTGCATCCTGGCCTGTGCCCCTCTGGCTTTGGTACTGGGATTTTTTTTTTCCTTTTATGTCATATTGATCCTGACACCATGGAACTTTTGGAGGTAGACAGGACCCACACATGGATTAGTTAAAAGCCTCCCATCCATCTAAGCTCATGGTAGGAGATAGAGCATGTCCAAGAGAGGAGGGCAGGCATCAGACCTAGAAGATATGGCTGGGCATCCAACCCAATCTCCCTTCCCGGAGAACAGACTCTAAGTCAGATCCAGCCACCCTTGAGTAACCAGCTCAAGGTACACAGAACAAGAGAGTCTGGTATACAGCAGGTGCTAAACAAATGCTTGTGGTAGCAAAAGCTATAGGTTTTGGGTCAGAACTCCGACCCAAGTCGCGACTGAAGAGCGAAAGGCCCTCTACTCGCCACCGCCCCGCCCCCACCTGGGGTCCTATAACAGATCACTTTCACCCTTGCGGGAGCCAGAGAGCCCTGGCATCCTAGGTAGCCCCCCCCGCCCCCCCCCCGCAAGCAGCCCAGCCCTGCCTTTGGGGCAAGTTCTTTTCTCAGCCTGGACCTGTGATAATGAGGGGGTTGGACGCGCCGCCTTTGGTCGCTTTCAAGTCTAATGAATTCTTATCCCTACCACCTGCCCTTCTACCCCGCGCCTCCAGAGCAGCTGTCCTGATTTATTACCTTCAATTAACCTCCACTCCTTTCTCCATCTCCTGGGATACCGCCCCTGTCCCAGTGGCTGGTAAAGGAGCTTAGGAAGGACCAGAGCCAGGTGTGGCTAGAGGCTACCAGGCAGGGCTGGGGATGAGGAGCTAAACTGGAAGAGTGTTTGGTTAGTAGGCACAAAGCCTTGGGTGGGATCCCTAGTACCGGAGAAGTGGAGATGGGCGCTGAGAAGTTCAAGACCATCCATCCTTAACTACACAGCCAGTTTGAGGCCAGCCTGGGCTACATAAAAACCCAATCTCAAAAGCTGCCAATTCTGATTCTGTGCCACGTAGTGCCCGATGTAATAGTGGATGAAGTCGTTGAATCCTGGGGCAACCTATTTTACAGATGTGGGGAAAAGCAACTTTAAGTACCCTGCCCACAGATCACAAAGAAAGTAAGTGACAGAGCTCCAGTGTTTCATCCCTGGGTTCCAAGGACAGGGAGAGAGAAGCCAGGGTGGGATCTCACTGCTCCCCGGTGCCTCCTTCCTATAATCCATACAGATTCGAAAGCGCAGGGCAGGTTTGGAAAAAGAGAGAAGGGTGGAAGGAGCAGACCAGTCTGGCCTAGGCTGCAGCCCCTCACGCATCCCTCTCTCCGCAGATGTGTCCGAGTACAGCTGCCGCGAGCTGCACTACACCCGCTTCCTGACAGACGGCCCATGCCGCAGCGCCAAGCCGGTCACCGAGTTGGTGTGCTCCGGCCAGTGCGGCCCCGCGCGGCGCTGCCCAACGCCATCGGGCGCGTGAAGGTGGTGGCGCCCGAACGGACCGGATTTCCGCTGCATCCCGGATCGCTACCGCGCGCAGCGGGTGCAGCTGCTGTGCCCCGGGGGCGCGGCGCCGCGCTCGCGCAAGGTGCGTCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACCCGCTTCCACAACCAGTCGGAGCTCAAGGACTTCGGGCCGGAGACCGCGCGGCCGCAGAAGGGTCGCAAGCCGCGGCCCGGCGCCCGGGGAGCCAAAGCCAACCAGGCGGAGCTGGAGAACGCCTACTAGAGCGAGCCCGCGCCTATGCAGCCCCCGCGCGATCGCATTCGTTTTCAGTGTAAAGCCTGCAGCCCAGGCCAGGGGTGCCAAACTTTCCAGACCGTGTGGAGTTCCCAGCCCAGTAGAGACCGCAGGTCCTTCTGCCCGCTGCGGGGGATGGGGAGGGGGTGGGGTTCCCGCGGGCCAGGAGAGGAAGCTTGAGTCCCAGACTCTGCCTAGCCCCGGGTGGGATGGGGGTCTTTCTACCCTCGCCGGACCTATACAGGACAAGGCAGTGTTTCCACCTTAAAGGGAAGGGAGTGTGGAACGAAAGACCTGGGACTGGTTATGGACGTACAGTAAGATCTACTCCTTCCACCCAAATGTAAAGCCTGCGTGGGCTAGATAGGGTTTCTGACCCTGACCTGGCCACTGAGTGTGATGTTGGGCTACGTGGTTCTCTTTTGGTACGGTCTTCTTTGTAAAATAGGGACCGGAACTCTGCTGAGATTCCAAGGATTGGGGTACCCCGTGTAGACTGGTGAGAGAGAGGAGAACAGGGGAGGGGTTAGGGGAGAGATTGTGGTGGGCAACCGCCTAGAAGAAGCTGTTTGTTGGCTCCCAGCCTCGCCGCCTCAGAGGTTTGGCTTCCCCCACTCCTTCCTCTCAAATCTGCCTTCAAATCCATATCTGGGATAGGGAAGGCCAGGGTCCGAGAGATGGTGGAAGGGCCAGAAATCACACTCCTGGCCCCCCGAAGAGCAGTGTCCCGCCCCCAACTGCCTTGTCATATTGTAAAGGGATTTTCTACACAACAGTTTAAGGTCGTTGGAGGAAACTGGGCTTGCCAGTCACCTCCCATCCTTGTCCCTTGCCAGGACACCACCTCCTGCCTGCCACCCACGGACACATTTCTGTCTAGAAACAGAGCGTCGTCGTGCTGTCCTCTGAGACAGCATATCTTACATTAAAAAGAATAATACGGGGGGGGGGGGCGGAGGGCGCAAGTGTTATACATATGCTGAGAAGCTGTCAGGCGCCACAGCACCACCCACAATCTTTTTGTAAATCATTTCCAGACACCTCTTACTTTCTGTGTAGATTTTAATTGTTAAAAGGGGAGGAGAGAGAGCGTTTGTAACAGAAGCACATGGAGGGGGGGGTAGGGGGGTTGGGGCTGGTGAGTTTGGCGAACTTTCCATGTGAGACTCATCCACAAAGACTGAAAGCCGCGTTTTTTTTTTTAAGAGTTCAGTGACATATTTATTTTCTCATTTAAGTTATTTATGCCAACATTTTTTTCTTGTAGAGAAAGGCAGTGTTAATATCGCTTTGTGAAGCACAAGTGTGTGTGGTTTTTTGTTTTTTGTTTTTTCCCCGACCAGAGGCATTGTTAATAAAGACAATGAATCTCGAGCAGGAGGCTGTGGTCTTGTTTTGTCAACCACACACAATGTCTCGCCACTGTCATCTCACTCCCTTCCCTTGGTCACAAGACCCAAACCTTGACAACACCTCCGACTGCTCTCTGGTAGCCCTTGTGGCAATACGTGTTTCCTTTGAAAAGTCACATTCATCCTTTCCTTTGCAAACCTGGCTCTCATTCCCCAGCTGGGTCATCGTCATACCCTCACCCCAGCCTCCCTTTAGCTGACCACTCTCCACACTGTCTTCCAAAAGTGCACGTTTCACCGAGCCAGTTCCCTGGTCCAGGTCATCCCATTGCTCCTCCTTGCTGGAGACCCTTCTCCCACAAAGATGTTCATCTCCCACTCCATCAAGCCCCAGTGGCCCTGCGGCTATCCCTGTCTCTTCAGTTAGCTGAATCTACTTGCTGACACCACATGAATTCCTTCCCCTGTCTTAAGGTTCATGGAACTCTTGCCTGCCCCTGAACCTTCCAGGACTGTCCCAGCGTCTGATGTGTCCTCTCTCTTGTAAAGCCCCACCCCACTATTTGATTCCCAATTCTAGATCTTCCCTTGTTCATTCCTTCACGGGATAGTGTCTCATCTGGCCAAGTCCTGCTTGATATTGGGATAAATGCAAAGCCAAGTACAATTGAGGACCAGTTCATCATTGGGCCAAGCTTTTTCAAAATGTGAATTTTACACCTATAGAAGTGTAAAAGCCTTCCAAAGCAGAGGCAATGCCTGGCTCTTCCTTCAACATCAGGGCTCCTGCTTTATGGGTCTGGTGGGGTAGTACATTCATAAACCCAACACTAGGGGTGTGAAAGCAAGATGATTGGGAGTTCGAGGCCAATCTTGGCTATGAGGCCCTGTCTCAACCTCTCCTCCCTCCCTCCAGGGTTTTGTTTTGTTTTGTTTTTTTGATTTGAAACTGCAACACTTTAAATCCAGTCAAGTGCATCTTTGCGTGAGGGGAACTCTATCCCTAATATAAGCTTCCATCTTGATTTGTGTATGTGCACACTGGGGGTTGAACCTGGGCCTTTGTACCTGCCGGGCAAGCTCTCTACTGCTCTAAACCCAGCCCTCACTGGCTTTCTGTTTCAACTCCCAATGAATTCCCCTAAATGAATTATCAATATCATGTCTTTGAAAAATACCATTGAGTGCTGCTGGTGTCCCTGTGGTTCCAGATTCCAGGAAGGACTTTTCAGGGAATCCAGGCATCCTGAAGAATGTCTTAGAGCAGGAGGCCATGGAGACCTTGGCCAGCCCCACAAGGCAGTGTGGTGCAGAGGGTGAGGATGGAGGCAGGCTTGCAATTGAAGCTGAGACAGGGTACTCAGGATTAAAAAGCTTCCCCCAAAACAATTCCAAGATCAGTTCCTGGTACTTGCACCTGTTCAGCTATGCAGAGCCCAGTGGGCATAGGTGAAGACACCGGTTGTACTGTCATGTACTAACTGTGCTTCAGAGCCGGCAGAGACAAATAATGTTATGGTGACCCCAGGGGACAGTGATTCCAGAAGGAACACAGAAGAGAGTGCTGCTAGAGGCTGCCTGAAGGAGAAGGGGTCCCAGACTCTCTAAGCAAAGACTCCACTCACATAAAGACACAGGCTGAGCAGAGCTGGCCGTGGATGCAGGGAGCCCATCCACCATCCTTTAGCATGCCCTTGTATTCCCATCACATGCCAGGGATGAGGGGCATCAGAGAGTCCAAGTGATGCCCAAACCCAAACACACCTAGGACTTGCTTTCTGGGACAGACAGATGCAGGAGAGACTAGGTTGGGCTGTGATCCCATTACCACAAAGAGGGAAAAAACAAAAAACAAACAAACAAACAAAAAAAAACAAAACAAAACAAAAAAAAACCCAAGGTCCAAATTGTAGGTCAGGTTAGAGTTTATTTATGGAAAGTTATATTCTACCTCCATGGGGTCTACAAGGCTGGCGCCCATCAGAAAGAACAAACAACAGGCTGATCTGGGAGGGGTGGTACTCTATGGCAGGGAGCACGTGTGCTTCCCCTACAGCCAGACACGGGGCTTGTATTAATCACAGGGCTTGTATTAATAGGCTGAGAGTCAAGCAGACAGAGAGACAGAAGGAAACACACACACACACACACACACACACACACACACACACACATGCACACACCACTCACTTCTCACTCGAAGAGCCCCTACTTACATTCTAAGAACAAACCATTCCTCCTCATAAAGGAGACAAAGTTGCAGAAACCCAAAAGAGCCACAGGGTCCCCACTCTCTTTGAAATGACTTGGACTTGTTGCAGGGAAGACAGAGGGGTCTGCAGAGGCTTCCTGGGTGACCCAGAGCCACAGACACTGAAATCTGGTGCTGAGACCTGTATAAACCCTCTTCCACAGGTTCCCTGAAAGGAGCCCACATTCCCCAACCCTGTCTCCTGACCACTGAGGATGAGAGCACTTGGGCCTTCCCCATTCTTGGAGTGCACCCTGGTTTCCCCATCTGAGGGCACATGAGGTCTCAGGTCTTGGGAAAGTTCCACAAGTATTGAAAGTGTTCTTGTTTTGTTTGTGATTTAATTTAGGTGTATGAGTGCTTTTGCTTGAATATATGCCTGTGTAGCATTTACAAGCCTGGTGCCTGAGGAGATCAGAAGATGGCATCAGATACCCTGGAACTGGACTTGCAGACAGTTATGAGCCACTGTGTGGGTGCTAGGAACAGAACCTGGATCCTCCGGAAGAGCAGACAGCCAGCGCTCTTAGCCACTAAGCCATCACTGAGGTTCTTTCTGTGGCTAAAGAGACAGGAGACAAAGGAGAGTTTCTTTTAGTCAATAGGACCATGAATGTTCCTCGTAACGTGAGACTAGGGCAGGGTGATCCCCCAGTGACACCGATGGCCCTGTGTAGTTATTAGCAGCTCTAGTCTTATTCCTTAATAAGTCCCAGTTTGGGGCAGGAGATATGTATTCCCTGCTTTGAAGTGGCTGAGGTCCAGTTATCTACTTCCAAGTACTTGTTTCTCTTTCTGGAGTTGGGGAAGCTCCCTGCCTGCCTGTAAATGTGTCCATTCTTCAACCTTAGACAAGATCACTTTCCCTGAGCAGTCAGGCCAGTCCAAAGCCCTTCAATTTAGCTTTCATAAGGAACACCCCTTTTGTTGGGTGGAGGTAGCACTTGCCTTGAATCCCAGCATTAAGAAGGCAGAGACAGTCGGATCTCTGTGAGTTCACAGCCAGCCTGGTCTACGGAGTGAGTTCCAAGACAGCCAGGCCTACACAGAGAAACCCTGTCTCGAAAAAAACAAAAACAAAAGAAATAAAGAAAAAGAAAACAAAAACGAACAAACAGAAAAACAAGCCAGAGTGTTTGTCCCCGTATTTTATTAATCATATTTTTGTCCCTTTGCCATTTTAGACTAAAAGACTCGGGAAAGCAGGTCTCTCTCTGTTTCTCATCCGGACACACCCAGAACCAGATGTATGGAAGATGGCTAATGTGCTGCAGTTGCACATCTGGGGCTGGGTGGATTGGTTAGATGGCATGGGCTGGGTGTGGTTACGATGACTGCAGGAGCAAGGAGTATGTGGTGCATAGCAAACGAGGAAGTTTGCACAGAACAACACTGTGTGTACTGATGTGCAGGTATGGGCACATGCAAGCAGAAGCCAAGGGACAGCCTTAGGGTAGTGTTTCCACAGACCCCTCCCCCCTTTTAACATGGGCATCTCTCATTGGCCTGGAGCTTGCCAACTGGGCTGGGCTGGCTAGCTTGTAGGTCCCAGGGATCTGCATATCTCTGCCTCCCTAGTGCTGGGATTACAGTCATATATGAGCACACCTGGCTTTTTTATGTGGGTTCTGGGCTTTGAACCCAGATCTGAGTGCTTGCAAGGCAATCGGTTGAATGACTGCTTCATCTCCCCAGACCCTGGGATTCTACTTTCTATTAAAGTATTTCTATTAAATCAATGAGCCCCTGCCCCTGCACTCAGCAGTTCTTAGGCCTGCTGAGAGTCAAGTGGGGAGTGAGAGCAAGCCTCGAGACCCCATCAGCGAAGCAGAGGACAAAGAAATGAAAACTTGGGATTCGAGGCTCGGGATATGGAGATACAGAAAGGGTCAGGGAAGGAAATGAACCAGATGAATAGAGGCAGGAAGGGTAGGGCCCTGCATACATGGAACCTGGTGTACATGTTATCTGCATGGGGTTTGCATTGCAATGGCTCTTCAGCAGGTTCACCACACTGGGAAACAGAAGCCAAAAAGAAGAGTAGGTGGTGTTGGAGTCAGATACTGTCAGTCATGCCTGAAGAAATGGAAGCAATTAACGATGCGCCGCAATTAGGATATTAGCTCCCTGAAGAAAGGCAAGAAGCTGGGCTGTGGGCACTGAAGGGAGCTTTGAATGATGTCACATTCTCTGTATGCCTAGCAGGGCAGTATTGGAGACTGAGACTTGACTTGTGTGTCCATATGATTCCTCCTTTTCCTACAGTCATCTGGGGCTCCTGAGCTTCGTCCTTGTCCAAGAACCTGGAGCTGGCAGTGGGCAGCTGCAGTGATAGATGTCTGCAAGAAAGATCTGAAAAGAGGGAGGAAGATGAAGGACCCAGAGGACCACCGACCTCTGCTGCCTGACAAAGCTGCAGGACCAGTCTCTCCTACAGATGGGAGACAGAGGCGAGAGATGAATGGTCAGGGGAGGAGTCAGAGAAAGGAGAGGGTGAGGCAGAGACCAAAGGAGGGAAACACTTGTGCTCTACAGCTACTGACTGAGTACCAGCTGCGTGGCAGACAGCCAATGCCAAGGCTCGGCTGATCATGGCACCTCGTGGGACTCCTAGCCCAGTGCTGGCAGAGGGGAGTGCTGAATGGTGCATGGTTTGGATATGATCTGAATGTGGTCCAGCCCTAGTTTCCTTCCAGTTGCTGGGATAAAGCACCCTGACCAAAGCTACTTTTTTGTTTGTTTGTTTTGGTTTGGTTTTGTTTGGTTTTTCGAGGCAGGGTTTCTCTGTATCACCCTAGCTGTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTCGAACTCAGAAATCCCCCTGCCTCTGCCTCCTAAGTGCTGGAATTAAAGGCCTGCGCCACCACTGCCGGCCCAAAGCTACTTTAAGAGAGAGAGAGGAATGTATAAGTATTATAATTCCAGGTTATAGTTCATTGCTGTAGAATTGGAGTCTTCATATTCCAGGTAATCTCCCACAGACATGCCACAAAACAACCTGTTCTACGAAATCTCTCATGGACTCCCTTCCCCAGTAATTCTAAACTGTGTCAAATCTACAAGAAATAGTGACAGTCACAGTCTCTAACGTTTTGGGCATGAGTCTGAAGTCTCATTGCTAAGTACTGGGAAGATGAAAACTTTACCTAGTGTCAGCATTTGGAGCAGAGCCTTTGGGATTTGAGATGGTCTTTTGCAGAGCTCCTAATGGCTACATGGAGAGAGGGGGCCTGGGAGAGACCCATACACCTTTTGCTGCCTTATGTCACCTGACCTGCTCCTTGGGAAGCTCTAGCAAGAAGGCCTTCCCTGGATCACCCACCACCTTGCACCTCCAGAACTCAGAGCCAAATTAAACTTTCTTGTTACTGTCGTCAAAGCACAGTCGGTCTGGGTTGTATCACTGTCAATGGGAAACAGACTTGCCTGGATGGATAACTTGTACATTGCATAATGTCTAGAAATGAAAAGTCCTATAGAGAAAAAGAAAATTAGCTGGCACACAGATAGAGGCCCTGGAGGAGGCTGGCTTTGTCCTCCCCGAGGAGGTGGCGAGTAAGGTGTAAATGTTCATGGATGTAAATGGGCCCATATATGAGGGTCTGGGGTAACAAGAAGGCCTGTGAATATAAAGCACTGAAGGTATGTCTAGTCTGGAGAAGGTCACTACAGAGAGTTCTCCAACTCAGTGCCCATACACACACACACACACACACACACACACACACACACACACACACACCACAAAGAAAAAAAGGAAGAAAAATCTGAGAGCAAGTACAGTACTTAAAATTGTGTGATTGTGTGTGTGACTCTGATGTCACATGCTCATCTTGCCCTATGAGTTGAAAACCAAATGGCCCCTGAGAGGCATAACAACCACACTGTTGGCTGTGTGCTCACGTTTTTCTTAAAGCGTCTGTCTGGTTTGCTGCTAGCATCAGGCAGACTTGCAGCAGACTACATATGCTCAGCCCTGAAGTCCTTCTAGGGTGCATGTCTCTTCAGAATTTCAGAAAGTCATCTGTGGCTCCAGGACCGCCTGCACTCTCCCTCTGCCGCGAGGCTGCAGACTCTAGGCTGGGGTGGAAGCAACGCTTACCTCTGGGACAAGTATAACATGTTGGCTTTTCTTTCCCTCTGTGGCTCCAACCTGGACATAAAATAGATGCAAGCTGTGTAATAAATATTTCCTCCCGTCCACTTAGTTCTCAACAATAACTACTCTGAGAGCACTTATTAATAGGTGGCTTAGACATAAGCTTTGGCTCATTCCCCCACTAGCTCTTACTTCTTTAACTCTTTCAAACCATTCTGTGTCTTCCACATGGTTAGTTACCTCTCCTTCCATCCTGGTTCGCTTCTTCCTTCGAGTCGCCCTCAGTGTCTCTAGGTGATGCTTGTAAGATATTCTTTCTACAAAGCTGAGAGTGGTGGCACTCTGGGAGTTCAAAGCCAGCCTGATCTACACAGCAAGCTCCAGGATATCCAGGGCAATGTTGGGAAAACCTTTCTCAAACAAAAAGAGGGGTTCAGTTGTCAGGAGGAGACCCATGGGTTAAGAAGTCTAGACGAGCCATGGTGATGCATACCTTTCATCCAAGCACTTAGGAGGCAAAGAAAGGTGAAACTCTTTGACTTTGAGGCCAGCTAGGTTACATAGTGATACCCTGCTTAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAATTTAAAAGTCTAAAAATGCATTCTTTTAAAAATATGTATAAGTATTTGCCTGCACATATGTATGTATGTATGTATACCATGTGTGTGTCTGGTGCTGAAGGACTAGGCATAGACTCCCTAGAACTAGAGTCATAGACAGTTGTGACACTCCCCAACCCCCCACCATGTGGGTGCTTGAAGCTAAACTCCTGTCCTTTGTAAAGCAGCAGGTGTCTATGAACCCTGAACCATCTCTCCAGTCTCCAGATGTGCATTCTCAAAGAGGAGTCCTTCATATTTCCCTAAACTGAACATCCTTATCAGTGAGCATCCTCGAGTCACCAAAGCTACTGCAAACCCTCTTAGGGAACATTCACTATTCACTTCTACTTGGCTCATGAAACTTAAGTACACACACACAAACACACACACACACACAGAGTCATGCACTCACAAAAGCATGCATGTACACCATTCTTATTAGACTATGCTTTGCTAAAAGACTTTCCTAGATACTTTAAAACATCACTTCTGCCTTTTGGTGGGCAGGTTCCAAGATTGGTACTGGCGTACTGGAAACTGAACAAGGTAGAGATCTAGAAATCACAGCAGGTCAGAAGGGCCAGCCTGTACAAGAGAGAGTTCCACACCTTCCAGGAACACTGAGCAGGGGGCTGGGACCTTGCCTCTCAGCCCAAGAAACTAGTGCGTTTCCTGTATGCATGCCTCTCAGAGATTCCATAAGATCTGCCTTCTGCCATAAGATCTCCTGCATCCAGACAAGCCTAGGGGAAGTTGAGAGGCTGCCTGAGTCTCTCCCACAGGCCCCTTCTTGCCTGGCAGTATTTTTTTATCTGGAGGAGAGGAATCAGGGTGGGAATGATCAAATACTATTATCAAGGAAAAAGTAAAAAACATATATATATATATATTAACTGATCTAGGGAGCTGGCTCAGCAGTTAAGAGTTCTGGCTGCCCTTGCTTCAGATCTTGCTTTGATTCCCAGCACCCACATGATGGCTTTCAACTGTATCTCTGCTTCCAGGGGATCCAACAGCCTCTTCTGACCTCCATAGACAAGACCTAGTCCTCTGCAAGAGCACCAAATGCTCTTATCTGTTGATCCATCTCTCTAGCCTCATGCCAGATCATTTAAAACTACTGGACACTGTCCCATTTTACGAAGATGTCACTGCCCAGTCATTTGCCATGAGTGGATATTTCGATTCTTTCTATGTTCTCACCCTTGCAATTTATAAGAAAGATATCTGCATTTGTCTCCTGAGAGAACAAAGGGTGGAGGGCTACTGAGATGGCTCTAGGGGTAAAGGTGCTTGCCACAAAATCTGACAACTTAAGTTTGGTCTTGGAATCCACATGGTGGAGAGAGAGAAGAGATTCCCGTAAGTTGTCCTCAAACTTCCCACACATGTGCTGTGGCTTATGTGTAACCCCAATAAGTAAAGATAGTTTTAAACACTACATAAGGTAGGGTTTCTTCATGACCCCAAGGAATGATGCCCCTGATAGAGCTTATGCTGAAACCCCATCTCCATTGTGCCATCTGGAAAGAGACAATTGCATCCCGGAAACAGAATCTTCATGAATGGATTAATGAGCTATTAAGAAAGTGGCTTGGTTATTGCACATGCTGGCGGCGTAATGACCTCCACCATGATGTTATCCAGCATGAAGGTCCTCACCAGAAGTCATACAAATCTTCTTAGGCTTCCAGAGTCGTGAGCAAAAAAAGCACACCTCTAAATAAATTAACTAGCCTCAGGTAGTTAACCACCGAAAATGAACCAAGGCAGTTCTAATACAAAACCACTTCCCTTCCCTGTTCAAACCACAGTGCCCTATTATCTAAAAGATAAACTTCAAGCCAAGCTTTTAGGTTGCCAGTATTTATGTAACAACAAGGCCCGTTGACACACATCTGTAACTCCTAGTACTGGGCCTCAGGGGCAGAGACAGGTGGAGCCCTGGAGTTTGAATTCCAGGTTCTGTGAGAAACTCTGTCTGAAAAGACAATATGGTGAGTGACCCGGGAGGATATCTGATATTGACTTCTGGCCAACACACAGCCATCTCTGCACATCTGTAGTTGCAAGCCTTTTGCACTAAGTTTGGCCAGAGTCAGAGTTTGCAAGTGTTTGTGGACTGAATGCACGTGTTGCTGGTGATCTACAAAGTCACCCTCCTTCTCAAGCTAGCAGCACTGGCTTCGGCCAGCTGCTCATTCAAGCCTCTTTGCAGAGTCATCACGGGGATGGGGGAGCAGGGCCCCTCCCTAGAACACCAAGCCTGTGGTTGTTTATTCAGGACATTATTGAGGGCCAAGATGACAGATAACTCTATCACTTGGCCAACAGTCGGGTGTTGCGGTGTTAGGTTATTTCTGTGTCTGCAGAAAACAGTGCAACCTGGACAAAAGAAATAAATGATATCATTTTTCATTCAGGCAACTAGATTCCGTGGTACAAAAGGCTCCCTGGGGAACGAGGCCGGGACAGCGCGGCTCCTGAGTCGCTATTTCCGTCTGTCAACTTCTCTAATCTCTTGATTTCCTCCCTCTGTCTGTTTCCTTCCTCTTGCTGGGGCCCAGTGGAGTCTGTGTACTCACAGGGAGGAGGGTGGCAAAGCCCTGGTCCTCTACGGGCTGGGGGAAGGGGGGAAGCTGTCGGCCCAGTGACTTTTTCCCCTTTCTCTTTTTCTTAGAAACCAGTCTCAATTTAAGATAATGAGTCTCCTCATTCACGTGTGCTCACTATTCATAGGGACTTATCCACCCCCGCCCTGTCAATCTGGCTAAGTAAGACAAGTCAAATTTAAAAGGGAACGTTTTTCTAAAAATGTGGCTGGACCGTGTGCCGGCACGAAACCAGGGATGGCGGTCTAAGTTACATGCTCTCTGCCAGCCCCGGTGCCTTTTCCTTTCGGAAAGGAGACCCGGAGGTAAAACGAAGTTGCCAACTTTTGATGATGGTGTGCGCCGGGTGACTCTTTAAAATGTCATCCATACCTGGGATAGGGAAGGCTCTTCAGGGAGTCATCTAGCCCTCCCTTCAGGAAAAGATTCCACTTCCGGTTTAGTTAGCTTCCACCTGGTCCCTTATCCGCTGTCTCTCCCCACTAGTCCTCATCCATCCGGTTTCCGCCCTCATCCACCTTGCCCTTTTAGTTCCTAGAAAGCAGCACCGTAGTCTTGGCAGGTGGGCCATTGGTCACTCCGCTACCACTGTTACCATGGCCACCAAGGTGTCATTTAAATATGAGCTCACTGAGTCCTGCGGGATGGCTTGGTTGGTAATATGCTTGCTGCAAAATCGTGAGAACTGGAGTTCAATTCCCAGCACATGGATGTATTTCCAGCACCTGGAAGGCAGGGAGCAGAGATCTTAAAGCTCCTGGCCAGACAGCCCAGCCTAATTAGTAATCAGTGAGAGACCCTGTCTCAAGAAACAAGATGGAACATCAAAGGTCAACCTCTTGTCTCCACACACACAAATACACACATGCACATACATCCACACACAGGCAAACACATGCACACACCTGAACACCCTCCACAAATACATACATAAAAAAATAAATACATACACACATACATACATACACCAACATTCCCTCTCCTTAGTCTCCTGGCTACGCTCTTGTCACCCCCACTAAGGCTTCAACTTCTTCTATTTCTTCATCTTGACTCCTCTGTACTTTGCATGCCTTTTCCAGCAAAGGCTTTTCTTTAAATCTCCGTCATTCATAAACTCCCTCTAAATTTCTTCCCCTGCCCTTTTCTTTCTCTCTAGGGAGATAAAGACACACACTACAAAGTCACCGTGGGACCAGTTTATTCACCCACCCACCCCTGCTTCTGTTCATCCGGCCAGCTAAGTAGTCCAACCTCTCTGGTGCTGTACCCTGGACCCTGGCTTCACCACAGCTCCTCCATGCTACCCAGCCCTGCAAACCTTCAGCCTAGCCTCTGGTTCTCCAACCAGCACAGGCCCAGTCTGGCTTCTATGTCCTAGAAATCTCCTTCATTCTCTCCATTTCCCTCCTGAATCTACCACCTTCTTTCTCCCTTCTCCTGACCTCTAATGTCTTGGTCAAACGATTACAAGGAAGCCAATGAAATTAGCAGTTTGGGGTACCTCAGAGTCAGCAGGGGAGCTGGGATGAATTCACATTTCCAGGCCTTTGCTTTGCTCCCCGGATTCTGACAGGCAGTTCCGAAGCTGAGTCCAGGAAGCTGAATTTAAAATCACACTCCAGCTGGGTTCTGAGGCAGCCCTACCACATCAGCTGGCCCTGACTGAGCTGTGTCTGGGTGGCAGTGGTGCTGGTGGTGCTGGTGGTGCTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGTGTGTGTGTGTTTTCTGCTTTTACAAAACTTTTCTAATTCTTATACAAAGGACAAATCTGCCTCATATAGGCAGAAAGATGACTTATGCCTATATAAGATATAAAGATGACTTTATGCCACTTATTAGCAATAGTTACTGTCAAAAGTAATTCTATTTATACACCCTTATACATGGTATTGCTTTTGTTGGAGACTCTAAAATCCAGATTATGTATTTAAAAAAAAATTCCCCAGTCCTTAAAAGGTGAAGAATGGACCCAGATAGAAGGTCACGGCACAAGTATGGAGTCGGAGTGTGGAGTCCTGCCAATGGTCTGGACAGAAGCATCCAGAGAGGGTCCAAGACAAATGCCTCGCCTCCTAAGGAACACTGGCAGCCCTGATGAGGTACCAGAGATTGCTAAGTGGAGGAATACAGGATCAGACCCATGGAGGGGCTTAAAGCGTGACTGTAGCAGCCCTCCGCTGAGGGGCTCCAGGTGGGCGCCCAAGGTGCTGCAGTGGGAGCCACATGAGAGGTGATGTCTTGGAGTCACCTCGGGTACCATTGTTTAGGGAGGTGGGGATTTGTGGTGTGGAGACAGGCAGCCTCAAGGATGCTTTTCAACAATGGTTGATGAGTTGGAACTAAAACAGGGGCCATCACACTGGCTCCCATAGCTCTGGGCTTGCCAGCTTCCACATCTGCCCCCCACCCCCTGTCTGGCACCAGCTCAAGCTCTGTGATTCTACACATCCAAAAGAGGAAGAGTAGCCTACTGGGCATGCCACCTCTTCTGGACCATCAGGTGAGAGTGTGGCAAGCCCTAGGCTCCTGTCCAGGATGCAGGGCTGCCAGATAGGATGCTCAGCTATCTCCTGAGCTGGAACTATTTTAGGAATAAGGATTATGCCCGCCCGGGGTTGGCCAGCACCCCAGCAGCCTGTGCTTGCGTAAAAGCAAGTGCTGTTGATTTATCTAAAAACAGAGCCGTGGACCCACCCACAGGACAAGTATGTATGCATCTGTTTCATGTATCTGAAAAGCGACACAACCATTTTTCACATCATGGCATCTTCCTAACCCCCATTCTTTTTTGTTTTGTTTTTTTGAGACAGGGTTTCTCTGTGTAGTCCTGGCTGTCCTGGAACTCACTTTGTAGACCAGGCTGGCCTCGAACTCAGAAATCCTGGGATTAAAGGTGTGTGCCACCACGCCCGGCCCTAACCCCCATTCTTAATGGTGATCCAGTGGTTGAAATTTCGGGCCACACACATGTCCATTAGGGATTAGCTGCTGTCTTCTGAGCTACCTGGTACAATCTTTATCCCCTGGGGCCTGGGCTCCTGATCCCTGACTCGGGCCCGATCAAGTCCAGTTCCTGGGCCCGATCAAGTCCAGTTCCTGGGCCCGAACAAGTCCAGTCCCTAGCTCGATTAGCTCATCCTGGCTCCCTGGCCTGTTCTTACTTACACTCTTCCCCTTGCTCTGGACTTGTTGCTTTCTTTACTCAAGTTGTCTGCCACAGTCCCTAAGCCACCTCTGTAAGACAACTAAGATAATACTTCCCTCAAGCACGGAAAGTCCTGAGTCACCACACCCTCTGGAGGTGTGTGGACACATGTTCATGCGTGTGGTTGCGCTTACGTACGTGTGCHuman Beer Genomic Sequence (This gene has two exons, at positions161–427 abd 3186–5219). Sequence ID No. 18 tagaggagaa gtctttggggagggtttgct ctgagcacac ccctttccct ccctccgggg 60 ctgagggaaa catgggaccagccctgcccc agcctgtcct cattggctgg catgaagcag 120 agaggggctt taaaaaggcgaccgtgtctc ggctggagac cagagcctgt gctactggaa 180 ggtggcgtgc cctcctctggctggtaccat gcagctccca ctggccctgt gtctcgtctg 240 cctgctggta cacacagccttccgtgtagt ggagggccag gggtggcagg cgttcaagaa 300 tgatgccacg gaaatcatccccgagctcgg agagtacccc gagcctccac cggagctgga 360 gaacaacaag accatgaaccgggcggagaa cggagggcgg cctccccacc acccctttga 420 gaccaaaggt atggggtggaggagagaatt cttagtaaaa gatcctgggg aggttttaga 480 aacttctctt tgggaggcttggaagactgg ggtagaccca gtgaagattg ctggcctctg 540 ccagcactgg tcgaggaacagtcttgcctg gaggtggggg aagaatggct cgctggtgca 600 gccttcaaat tcaggtgcagaggcatgagg caacagacgc tggtgagagc ccagggcagg 660 gaggacgctg gggtggtgagggtatggcat cagggcatca gaacaggctc aggggctcag 720 aaaagaaaag gtttcaaagaatctcctcct gggaatatag gagccacgtc cagctgctgg 780 taccactggg aagggaacaaggtaaggg&g cctcccatcc acagaacagc acctgtgggg 840 caccggacac tctatgctggtggtggctgt ccccaccaca cagacccaca tcatggaatc 900 cccaggaggt gaacccccagctcgaagggg aagaaacagg ttccaggcac tcagtaactt 960 ggtagtgaga agagctgaggtgtgaacctg gtttgatcca actgcaagat agccctggtg 1020 tgtggggggg tgtgggggacagatctccac aaagcagtgg ggaggaaggc cagagaggca 1080 cccctgcagt gtgcattgcccatggcctgc ccagggagct ggcacttgaa ggaatgggag 1140 ttttcggcac agttttagcccctgacatgg gtgcagctga gtccaggccc tggaggggag 1200 agcagcatcc tctgtgcaggagtagggaca tctgtcctca gcagccaccc cagtcccaac 1260 cttgcctcat tccaggggagggagaaggaa gaggaaccct gggttcctgg tcaggcctgc 1320 acagagaagc ccaggtgacagtgtgcatct ggctctataa ttggcaggaa tcctgaggcc 1380 atgggggcgt ctgaaatgacacttcagact aagagcttcc ctgtcctctg gccattatcc 1440 aggtggcaga gaagtccactgcccaggctc ctggacccca gccctccccg cctcacaacc 1500 tgttgggact atggggtgctaaaaagggca actgcatggg aggccagcca ggaccctccg 1560 tcttcaaaat ggaggacaagggcgcctccc cccacagctc cccttctagg caaggtcagc 1620 tgggctccag cgactgcctgaagggctgta aggaacccaa acacaaaatg tccaccttgc 1680 tggactccca cgagaggccacagcccctga ggaagccaca tgctcaaaac aaagtcatga 1740 tctgcagagg aagtgcctggcctaggggcg ctattctcga aaagccgcaa aatgccccct 1800 tccctgggca aatgcccccctgaccacaca cacattccag ccctgcagag gtgaggatgc 1860 aaaccagccc acagaccagaaagcagcccc agacgatggc agtggccaca tctcccctgc 1920 tgtgcttgct cttcagagtgggggtggggg gtggccttct ctgtcccctc tctggtttgg 1980 tcttaagact atttttcattctttcttgtc acattggaac tatccccatg aaacctttgg 2040 gggtggactg gtactcacacgacgaccagc tatttaaaaa gctcccaccc atctaagtcc 2100 accataggag acatggtcaaggtgtgtgca ggggatcagg ccaggcctcg gagcccaatc 2160 tctgcctgcc cagggagtatcaccatgagg cgcccattca gataacacag aacaagaaat 2220 gtgcccagca gagagccaggtcaatgtttg tggcagctga acctgtaggt tttgggtcag 2280 agctcagggc ccctatggtaggaaagtaac gacagtaaaa agcagccctc agctccatcc 2340 cccagcccag cctcccatggatgctcgaac gcagagcctc cactcttgcc ggagccaaaa 2400 ggtgctggga ccccagggaagtggagtccg gagatgcagc ccagcctttt gggcaagttc 2460 ttttctctgg ctgggcctcagtattctcat tgataatgag ggggttggac acactgcctt 2520 tgattccttt caagtctaatgaattcctgt cctgatcacc tccccttcag tccctcgcct 2580 ccacagcagc tgccctgatttattaccttc aattaacctc tactcctttc tccatcccct 2640 gtccacccct cccaagtggctggaaaagga atttgggaga agccagagcc aggcagaagg 2700 tgtgctgagt acttaccctgcccaggccag ggaccctgcg gcacaagtgt ggcttaaatc 2760 ataagaagac cccagaagagaaatgataat aataatacat aacagccgac gctttcagct 2820 atatgtgcca aatggtattttctgcattgc gtgtgtaatg gattaactcg caatgcttgg 2880 ggcggcccat tttgcagacaggaagaagag agaggttaag gaacttgccc aagatgacac 2940 ctgcagtgag cgatggagccctggtgtttg aaccccagca gtcatttggc tccgagggga 3000 cagggtgcgc aggagagctttccaccagct ctagagcatc tgggaccttc ctgcaataga 3060 tgttcagggg caaaagcctctggagacagg cttggcaaaa gcagggctgg ggtggagaga 3120 gacgggccgg tccagggcaggggtggccag gcgggcggcc accctcacgc gcgcctctct 3180 ccacagacgt gtccgagtacagctgccgcg agctgcactt cacccgctac gtgaccgatg 3240 ggccgtgccg cagcgccaagccggtcaccg agctggtgtg ctccggccag tgcggcccgg 3300 cgcgcctgct gcccaacgccatcggccgcg gcaagtggtg gcgacctagt gggcccgact 3360 tccgctgcat ccccgaccgctaccgcgcgc agcgcgtgca gctgctgtgt cccggtggtg 3420 aggcgccgcg cgcgcgcaaggtgcgcctgg tggcctcgtg caagtgcaag cgcctcaccc 3480 gcttccacaa ccagtcggagctcaaggact tcgggaccga ggccgctcgg ccgcagaagg 3540 gccggaagcc gcggccccgcgcccggagcg ccaaagccaa ccaggccgag ctggagaacg 3600 cctactagag cccgcccgcgcccctcccca ccggcgggcg ccccggccct gaacccgcgc 3660 cccacatttc tgtcctctgcgcgtggtttg attgtttata tttcattgta aatgcctgca 3720 acccagggca gggggctgagaccttccagg ccctgaggaa tcccgggcgc cggcaaggcc 3780 cccctcagcc cgccagctgaggggtcccac ggggcagggg agggaattga gagtcacaga 3840 cactgagcca cgcagccccgcctctggggc cgcctacctt tgctggtccc acttcagagg 3900 aggcagaaat ggaagcattttcaccgccct ggggttttaa gggagcggtg tgggagtggg 3960 aaagtccagg gactggttaagaaagttgga taagattccc ccttgcacct cgctgcccat 4020 cagaaagcct gaggcgtgcccagagcacaa gactgggggc aactgtagat gtggtttcta 4080 gtcctggctc tgccactaacttgctgtgta accttgaact acacaattct ccttcgggac 4140 ctcaatttcc actttgtaaaatgagggtgg aggtgggaat aggatctcga ggagactatt 4200 ggcatatgat tccaaggactccagtgcctt ttgaatgggc agaggtgaga gagagagaga 4260 gaaagagaga gaatgaatgcagttgcattg attcagtgcc aaggtcactt ccagaattca 4320 gagttgtgat gctctcttctgacagccaaa gatgaaaaac aaacagaaaa aaaaaagtaa 4380 agagtctatt tatggctgacatatttacgg ctgacaaact cctggaagaa gctatgctgc 4440 ttcccagcct ggcttccccggatgtttggc tacctccacc cctccatctc aaagaaataa 4500 catcatccat tggggtagaaaaggagaggg tccgagggtg gtgggaggga tagaaatcac 4560 atccgcccca acttcccaaagagcagcatc cctcccccga cccatagcca tgttttaaag 4620 tcaccttccg aagagaagtgaaaggttcaa ggacactggc cttgcaggcc cgagggagca 4680 gccatcacaa actcacagaccagcacatcc cttttgagac accgccttct gcccaccact 4740 cacggacaca tttctgcctagaaaacagct tcttactgct cttacatgtg atggcatatc 4800 ttacactaaa agaatattattgggggaaaa actacaagtg ctgtacatat gctgagaaac 4860 tgcagagcat aatagctgccacccaaaaat ctttttgaaa atcatttcca gacaacctct 4920 tactttctgt gtagtttttaattgttaaaa aaaaaaagtt ttaaacagaa gcacatgaca 4980 tatgaaagcc tgcaggactggtcgtttttt tggcaattct tccacgtggg acttgtccac 5040 aagaatgaaa gtagtggtttttaaagagtt aagttacata tttattttct cacttaagtt 5100 atttatgcaa aagtttttcttgtagagaat gacaatgtta atattgcttt atgaattaac 5160 agtctgttct tccagagtccagagacattg ttaataaaga caatgaatca tgaccgaaag 5220 gatgtggtct cattttgtcaaccacacatg acgtcatttc tgtcaaagtt gacacccttc 5280 tcttggtcac tagagctccaaccttggaca cacctttgac tgctctctgg tggcccttgt 5340 ggcaattatg tcttcctttgaaaagtcatg tttatccctt cctttccaaa cccagaccgc 5400 atttcttcac ccagggcatggtaataacct cagccttgta tccttttagc agcctcccct 5460 ccatgctggc ttccaaaatgctgttctcat tgtatcactc ccctgctcaa aagccttcca 5520 tagctccccc ttgcccaggatcaagtgcag tttccctatc tgacatggga ggccttctct 5580 gcttgactcc cacctcccactccaccaagc ttcctactga ctccaaatgg tcatgcagat 5640 ccctgcttcc ttagtttgccatccacactt agcaccccca ataactaatc ctctttcttt 5700 aggattcaca ttacttgtcatctcttcccc taaccttcca gagatgttcc aatctcccat 5760 gatccctctc tcctctgaggttccagcccc ttttgtctac accactactt tggttcctaa 5820 ttctgttttc catttgacagtcattcatgg aggaccagcc tggccaagtc ctgcttagta 5880 ctggcataga caacacaaagccaagtacaa ttcaggacca gctcacagga aacttcatct 5940 tcttcgaagt gtggatttgatgcctcctgg gtagaaatgt aggatdttca aaagtgggcc 6000 agcctcctgc acttctctcaaagtctcgcc tccccaaggt gtcttaatag tgctggatgc 6060 tagctgagtt agcatcttcagatgaagagt aaccctaaag ttactcttca gttgccctaa 6120 ggtgggatgg tcaactggaaagctttaaat taagtccagc ctaccttggg ggaacccacc 6180 cccacaaaga aagctgaggtccctcctgat gacttgtcag tttaactacc aataacccac 6240 ttgaattaat catcatcatcaagtctttga taggtgtgag tgggtatcag tggccggtcc 6300 cttcctgggg ctccagcccccgaggaggcc tcagtgagcc cctgcagaaa atccatgcat 6360 catgagtgtc tcagggcccagaatatgaga gcaggtagga aacagagaca tcttccatcc 6420 ctgagaggca gtgcggtccagtgggtgggg acacgggctc tgggtcaggt ttgtgttgtt 6480 tgtttgtttg ttttgagacagagtctcgct ctattgccca ggctggagtg cagtgtcaca 6540 atctcggctt actgcaacttctgccttccc ggattcaagt gattctcctg cctcagcctc 6600 cagagtagct gggattacaggtgcgtgcca ccacgcctgg ctaatttttg tatttttgat 6660 agagacgggg tttcaccatgttggccaggc tagtctcgaa ctcttgacct caagtgatct 6720 gcctgcctcg gcctcccaaagtgctgggat tacaggcgtg agccaccaca cccagcccca 6780 ggttggtgtt tgaatctgaggagactgaag caccaagggg ttaaatgttt tgcccacagc 6840 catacttggg ctcagttccttgccctaccc ctcacttgag ctgcttagaa cctggtgggc 6900 acatgggcaa taaccaggtcacactgtttt gtaccaagtg ttatgggaat ccaagatagg 6960 agtaatttgc tctgtggaggggatgaggga tagtggttag ggaaagcttc acaaagtggg 7020 tgttgcttag agattttccaggtggagaag ggggcttcta ggcagaaggc atagcccaag 7080 caaagactgc aagtgcatggctgctcatgg gtagaagaga atccaccatt cctcaacatg 7140 taccgagtcc ttgccatgtgcaaggcaaca tgggggtacc aggaattcca agcaatgtcc 7200 aaacctaggg tctgctttctgggacctgaa gatacaggat ggatcagccc aggctgcaat 7260 cccattacca cgagggggaaaaaaacctga aggctaaatt gtaggtcggg ttagaggtta 7320 tttatggaaa gttatattctacctacatgg ggtctataag cctggcgcca atcagaaaag 7380 gaacaaacaa cagacctagctgggaggggc agcattttgt tgtagggggc ggggcacatg 7440 ttctgggggt acagccagactcagggcttg tattaatagt ctgagagtaa gacagacaga 7500 gggatagaag gaaataggtccctttctctc tctctctctc tctctctctc actctctctc 7560 tctctcacac acacacacagacacacacac acgctctgta ggggtctact tatgctccaa 7620 gtacaaatca ggccacatttacacaaggag gtaaaggaaa agaacgttgg aggagccaca 7680 ggaccccaaa attccctgttttccttgaat caggcaggac ttacgcagct gggagggtgg 7740 agagcctgca gaagccacctgcgagtaagc caagttcaga gtcacagaca ccaaaagctg 7800 gtgccatgtc ccacacccgcccacctccca cctgctcctt gacacagccc tgtgctccac 7860 aacccggctc ccagatcattgattatagct ctggggcctg caccgtcctt cctgccacat 7920 ccccacccca ttcttggaacctgccctctg tcttctccct tgtccaaggg caggcaaggg 7980 ctcagctatt gggcagctttgaccaacagc tgaggctcct tttgtggctg gagatgcagg 8040 aggcagggga atattcctcttagtcaatgc gaccatgtgc ctggtttgcc cagggtggtc 8100 tcgtttacac ctgtaggccaagcgtaatta ttaacagctc ccacttctac tctaaaaaat 8160 gacccaatct gggcagtaaattatatggtg cccatgctat taagagctgc aacttgctgg 8220 gcgtggtggc tcacacctgtaatcccagta ctttgggacg tcaaggcggg tggatcacct 8280 gaggtcacga gttagagactggcctggcca gcatggcaaa accccatctt tactaaaaat 8340 acaaaaatta gcaaggcatggtggcatgca cctgtaatcc caggtactcg ggaggctgag 8400 acaggagaat ggcttgaacccaggaggcag aggttgcagt gagccaagat tgtgccactg 8460 ccctccagcc ctggcaacagagcaagactt catctcaaaa gaaaaaggat actgtcaatc 8520 actgcaggaa gaacccaggtaatgaatgag gagaagagag gggctgagtc accatagtgg 8580 cagcaccgac tcctgcaggaaaggcgagac actgggtcat gggtactgaa gggtgccctg 8640 aatgacgttc tgctttagagaccgaacctg agccctgaaa gtgcatgcct gttcatgggt 8700 gagagactaa attcatcattccttggcagg tactgaatcc tttcttacgg ctgccctcca 8760 atgcccaatt tccctacaattgtctggggt gcctaagctt ctgcccacca agagggccag 8820 agctggcagc gagcagctgcaggtaggaga gataggtacc cataagggag gtgggaaaga 8880 gagatggaag gagaggggtgcagagcacac acctcccctg cctgacaact tcctgagggc 8940 tggtcatgcc agcagatttaaggcggaggc aggggagatg gggcgggaga ggaagtgaaa 9000 aaggagaggg tggggatggagaggaagaga gggtgatcat tcattcattc cattgctact 9060 gactggatgc cagatgtgagccaggcacca ccctagctct gggcatgtgg ttgtaatctt 9120 ggagcctcat ggagctcacagggagtgctg gcaaggagat ggataatgga cggataacaa 9180 ataaacattt agtacaatgtccgggaatgg aaagttctcg aaagaaaaat aaagctggtg 9240 agcatataga cagccctgaaggcggccagg ccaggcattt ctgaggaggt ggcatttgag 9300 c 9301

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for altering bone mineral density, comprising administeringto an individual a therapeutically effective amount of an antibody thatbinds to a BEER polypeptide and increases bone density wherein the BEERpolypeptide is selected from the group consisting of: (a) a polypeptideencoded by a polynucleotide that comprises a nucleotide sequenceselected from SEQ ID NOs:1, 5, 9, 11, 13, and 15, and (b) a polypeptidethat comprises an amino acid sequence selected from SEQ ID NOs: 2, 6,10, 12, 14, and
 16. 2. The method of claim 1 wherein the antibody isselected from the group consisting of a polyclonal antibody, amonoclonal antibody, a humanized antibody, F(ab′)2, F(ab)₂, Fab′, Fab,and Fv.
 3. A method for treating a condition which results in loss ofbone mineral content, comprising administering to an individual atherapeutically effective amount of an antibody that binds to a BEERpolypeptide and increases bone density wherein the BEER polypeptide isselected from the group consisting of: (a) a polypeptide encoded by apolynucleotide that comprises a nucleotide sequence selected from SEQ IDNOs:1, 5, 9, 11, 13, and 15, and (b) a polypeptide that comprises anamino acid sequence selected from SEQ ID NOs: 2, 6, 10, 12, 14, and 16,wherein bone mineral content is increased.
 4. The method of claim 3wherein the condition which results in loss of bone mineral content isselected from the group consisting of osteopenia and osteoporosis. 5.The method of claim 3 wherein the condition which results in loss ofbone mineral content is selected from the group consisting of bonedysplasia and bone fracture.